Inducible mutagenesis of target genes

ABSTRACT

The present invention relates generally to mutagenesis of target genes that takes advantage of the natural mutagenic capabilities of B cells, and enhances those capabilities by bringing the process of diversification under control. The invention provides a method for rapidly and inducibly generating point mutations and other types of diversification in expressed genes, such as antibody genes. This method can be coupled with selection to identify B cell clones that produce, for example, antibodies of high affinity or specificity. The diversification process can be modulated, accelerated, halted, switched between methods of mutagenesis and the like. The modulation of diversification in accordance with the invention is both inducible and reversible. The invention provides a means of rapid and feasible development of a repertoire of variant immunoglobulins and other polypeptides.

This application claims benefit of U.S. provisional patent applicationNo. 60/932,672, filed May 31, 2007, the entire contents of which areincorporated by reference into this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under R01 GM041712,awarded by the National Institutes of Health. The U.S. government hascertain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to mutagenesis of target genesthat takes advantage of the natural mutagenic capabilities of B cells,and enhances those capabilities by bringing the process ofdiversification under control. The invention provides a method forrapidly and inducibly generating point mutations and other types ofdiversification in expressed genes, such as antibody genes. This methodcan be coupled with selection to identify B cell clones that produce,for example, antibodies of high affinity or specificity. Thediversification process can be modulated, accelerated, halted, switchedbetween methods of mutagenesis and the like. The modulation ofdiversification in accordance with the invention is both inducible andreversible. The invention provides a means of rapid and feasibledevelopment of a repertoire of variant immunoglobulins and otherpolypeptides.

BACKGROUND OF THE INVENTION

Antibodies are molecules that provide a key defense against infection inhumans. They are used as therapeutics in treatment of a variety ofdiseases, from infectious disease to cancer. They are also used asdiagnostic reagents in a huge variety of tests carried out daily inclinical and research laboratories.

Antibody specificity and affinity are modified in vivo by processes ofmutation, targeted to specific regions within the genes that encodeantibodies. Antibodies are encoded by two genes, referred to as theimmunoglobulin (Ig) heavy and light chain genes. The heavy and lightchains of polypeptides encoded by the Ig genes interact to form atetrameric molecule which is expressed on the cell surface as areceptor. Antibody molecules are bifunctional: one domain recognizesantigen, the other promotes removal of antigen from the body. Therecognition domain, referred to as the variable (V) region, is createdupon interaction of the heavy and light chain polypeptides in naturalantibodies. It is in fact variable in sequence from one antibody toanother. Variability in V region primary sequence (and hencethree-dimensional structure and antigen specificity) is the result ofprocesses which alter V region sequence by causing irreversible geneticchanges. These changes are programmed during B cell development, and canalso be induced in the body in response to environmental signals thatactivate B cells. Several genetic mechanisms contribute to thisvariability. Two subpathways of the same mechanism lead to two differentmutagenic outcomes, referred to as somatic hypermutation and geneconversion (reviewed (Maizels, 2005)). Somatic hypermutation insertspoint mutations. Somatic hypermutation provides the advantage ofenabling essentially any mutation to be produced, so a collection ofmutated V regions has essentially sampled a large variety of possiblemutations. Gene conversion inserts mutations that are “templated” byrelated but non-identical sequences. Gene conversion provides theadvantage of creating a repertoire based on information already storedin the genome, which may be optimized by evolutionary selection.

The modified antigen receptor is the target for selection. Ig isexpressed on the cell surface, which permits clonal selection of B cellswith desired specificity or affinity in a physiological context orwithin cultured cells. Cell surface Ig can readily be detected by flowcytometry of cells stained with anti-Ig. Binding of Ig molecules tospecific compounds can be detected as interaction with fluorescentderivatives of those compounds, analyzed by flow cytometry; and B cellsthat bind to specific compounds can also be recovered upon sorting byflow cytometry. B cells that bind to specific compounds can also beselected on solid supports carrying those compounds. Conversely, bindingto solid support also permits removal of B cells with unwanted bindingspecificities. Repetitive cycles of binding and release permitenrichment for high affinity binding.

Mutation and gene targeting occur in the DT40 B cell line. DT40 is achicken B cell line that proliferates readily in culture. DT40 has beenwidely documented to carry out constitutive mutagenesis of its heavy andlight chain immunoglobulin genes (Reynaud et al., 1987; Thompson andNeiman, 1987; Reynaud et al., 1989). Mutagenesis is targeted to the Vregions. Mutations are normally templated by copying relatednon-functional V gene segments, called “pseudo-V” genes, which arepresent in a linear array upstream of each functional V region.Templating is evident as tracts of sequence shared between the mutated Vtarget and one of the pseudo-V genes. Genetic alterations of DT40 havebeen shown to cause a switch from templated mutagenesis to nontemplatedmutagenesis, which results in somatic hypermutation, by a pathwayessentially identical to somatic hypermutation that alters the sequencesof V regions in human B cells (Sale et al., 2001). DT40 has also beenwidely documented to support very efficient homologous recombination, orgene targeting (Buerstedde et al., 2002; Sale, 2004). This enablescreation of derivatives in which specific genes or genomic regions havebeen modified or ablated; or in which one genetic region has beenreplaced with another.

Due to the limitations and challenges posed by currently availableapproaches to targeted mutagenesis there is a need in the art for thedevelopment of alternative methods and constructs. The present inventionfulfills this need, and further provides other related advantages.

SUMMARY OF THE INVENTION

The invention meets these needs and others by providing materials andmethods for diversification of target sequences. The invention providesa B cell modified to permit reversible induction of diversification of atarget gene. The cell comprises a cis-regulatory element operably linkedto a target gene of interest. A factor that modulates diversificationcan then be fused to a tethering factor that binds to the cis-regulatoryelement, thereby tethering the diversification factor to the region thatcontrols expression of the target gene. The B cell can be a chicken DT40B cell or other vertebrate B cell, with a human B cell or a chicken DT40B cell containing humanized immunoglobulin (Ig) genes (in which humanIgH and IgL replace chicken IgH and IgL) preferred for some embodiments.

Typically, the target gene comprises a promoter and a coding region. Inone embodiment, the target gene comprises an immunoglobulin (Ig) gene,wherein the Ig gene comprises an Ig gene enhancer and coding region. TheIg gene can be all or part of an IgL and/or IgH gene. The coding regioncan be native to the Ig gene, or a heterologous gene. In someembodiments, the target gene is or contains a non-Ig target domain fordiversification, as well as domains permitting display of the geneproduct on the B cell surface, including a transmembrane domain and acytoplasmic tail. The coding region of the target gene in the B cell ofthe invention need not comprise a complete coding region. In someembodiments, a particular region or domain is targeted fordiversification, and the coding region may optionally encode only aportion that includes the region or domain of interest.

The cis-regulatory element provides a landing pad in the region thatcontrols diversification and/or expression of the target gene. Thislanding pad is a place to which a tethering factor can bind in asequence-specific manner to this region of the DNA. In a typicalembodiment, the cis-regulatory element is a polymerized Lactose operator(LacO). In one embodiment, the element comprises about 80-100 repeats ofLacO. In another embodiment, the cis-regulatory element is a single ormultimerized tetracycline operator (TetO). A variety of molecules can beused as a cis-regulatory element, so long as the element serves thelanding pad function of providing a place to which a tethering factor (asequence-specific DNA binding protein) can bind to the DNA and bring adiversification factor, fused to the tethering factor, into sufficientproximity of the coding region so that diversification of the codingregion is capable of reversible regulation.

A tethering factor is one that binds to the cis-regulatory element in asequence-specific manner. In the embodiments in which LacO serves as acis-regulatory element, the Lac repressor, LacI, can serve as thetethering factor, and its binding to the cis-regulatory element, LacO,can be regulated by isopropyl-β-D-thio-galactoside (IPTG). In theabsence of IPTG, LacI binds LacO and diversification is accelerated (orotherwise regulated) by the presence of the diversification factor. IPTGcan be added in the event that a halt or reduction in activity of thediversification factor is desired. In embodiments in which TetO servesas the cis-regulatory element, TetR can be a suitable tethering factor,and the activity of the diversification factor can be regulated bytetracycline or doxycycline.

In some embodiments, the diversification factor is a transcriptionalregulator, a heterochromatin-associated protein, a histone chaperone, achromatin remodeler, a component of the nuclear pore complex, a generegulator or a combination thereof. Other molecules that can serve as adiversification factor include, but are not limited to, a DNA repairfactor, a DNA replication factor, a resolvase, a helicase, a cell cycleregulator, a ubquitylation factor, a sumoylation factor, or acombination thereof. In one embodiment, the transcriptional regulator isVP16 or E47. A typical heterochromatin-associated protein for use as adiversification factor is HP1. A representative histone chaperone isHIRA.

Also provided is a method of producing a repertoire of polypeptideshaving variant sequences of a polypeptide of interest viadiversification of polynucleotide sequences that encode the polypeptide.Typically, the method comprises culturing the B cell of the invention inconditions that allow expression of the diversification factor, whereinthe target gene of the B cell contains the coding region of thepolypeptide of interest, thereby permitting diversification of thecoding region. The method can further comprise maintaining the cultureunder conditions that permit proliferation of the B cell until aplurality of variant polypeptides and the desired repertoire isobtained.

In another embodiment, the invention provides a method of producing Bcells that produce an optimized polypeptide of interest. The methodcomprises culturing a B cell of the invention in conditions that allowexpression of the diversification factor, wherein the target gene of theB cell contains the coding region of the polypeptide of interest, andwherein the B cell expresses the polypeptide of interest on its surface.The method further comprises selecting cells from the culture thatexpress the polypeptide of interest on the B cell surface by selectingcells that bind a ligand that specifically binds the polypeptide ofinterest. These steps of culturing and selecting can be repeated untilcells are selected that have a desired affinity for the ligand thatspecifically binds the polypeptide of interest. In embodiments in whichthe polypeptide of interest is an Ig, such as an IgL, IgH or both, theligand may be a polypeptide, produced by recombinant or other means,that represents an antigen. The ligand can be bound to or linked to asolid support to facilitate selection, for example, bymagnetic-activated cell selection (MACS). In another example, the ligandcan be bound to or linked to a fluorescent tag, to allow for orfluorescence-activated cell sorting (FACS).

The methods of the invention can further comprise adding a regulatorymolecule to the culture, wherein the regulatory molecule modulatesbinding of the tethering factor to the cis-regulatory element, therebymodulating diversification of the coding region. In the examplesdiscussed above, IPTG, tetracycline and doxycycline serve as theregulatory molecules. Those skilled in the art are aware of otherregulatory molecules that can be used with a particular tethering factorto regulate diversification activity.

The regulatory molecule can modulate the diversification of the codingregion in a variety of ways. For example, in some embodiments, theregulatory molecule is added to the culture to effect modulation, andthe modulation can result in reducing or halting diversification, or inenhancing or accelerating diversification, depending on whether theparticular regulatory molecule is one that increases or decreasesbinding of the tethering factor to the cis-regulatory element, and onwhether that particular change in binding has the effect of increasingor decreasing diversification activity. In other embodiments, themodulation, be it reduction, halt or enhancement or acceleration of themodulation, is effected by removing or eliminating the regulatorymolecule from the culture. Likewise, modulation of diversification canbe effected by adding a gene to or eliminating a gene from the B cell,or by increasing or diminishing the expression of a gene in the B cell.One skilled in the art can readily appreciate all of the availablepermutations set forth above, each of which has the effect of alteringthe level or presence of a regulatory molecule in the B cell, and inturn, altering the tethering of the diversification factor to thecis-regulatory element and thereby altering the diversificationactivity.

Also provided is a kit that can be used to carry out the methods of theinvention. The kit comprises a B cell of the invention and fusionconstructs that express the corresponding tethering and diversificationfactors. For example, the B cell comprises a cis-regulatory elementoperably linked to a target gene, wherein the target gene comprises apromoter and a coding region. The kit further comprises one or morecontainers, with one or more fusion constructs stored in the containers.Each fusion construct comprises a polynucleotide that can be expressedin the B cell and that encodes a tethering factor fused to adiversification factor, wherein the tethering factor specifically bindsto the cis-regulatory element of the B cell. The B cell can include aplurality of cis-regulatory elements for use with a plurality of fusionconstructs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic illustration of antibody somatic hypermutation. Upperpanel: the complementarity-determining regions (CDRs) of an Ig moleculecontact antigen. Lower panel: mutations are concentrated at the genomicregions encoding the CDRs.

FIG. 2. Bar graph showing the effect of tethered cis-regulators on Iggene diversification in

DT40 PolyLacO λ cells. Clonal diversification rates were compared in thepresence and absence of IPTG.

FIG. 3. Schematic illustration of how antibody specificities evolveduring expansion of a B cell clone. Antibody is expressed on the cellsurface. Selection of a population that initially expresses multiplespecificities (top left) successively enriches for a desired specificity(thin double circles, center) and higher affinity (thick circles, bottomright).

FIG. 4A-B. Demonstration that E2A associates with the rearranged Igλlocus. FIG. 4A: Schematic of the rearranged and unrearrranged Igλ lociin the chicken B cell lymphoma line, DT40. Shown are promoter (P),leader (L), variable (V), joining (J) and constant (Cλ) regions; theputative matrix attachment region (M) in the J-C intron; the 3′-enhancer(E-3′); and the most proximal (ΨV1) and distal (ΨV25) of the upstreamnonfunctional pseudo-variable regions which are templates for geneconversion. The rearranged and unrearranged alleles can readily bedistinguished by PCR, using primers indicated by arrows. FIG. 4B: ChIPanalysis of E2A enrichment at the rearranged λR and unrearranged λU lociin DT40 cells, relative to ovalbumin gene control amplicon (Ova). Foldenrichment is shown below. NTC, no template control.

FIG. 5A-F. E2A acts in cis to regulate Igλ gene diversification. FIG.5A: Schematic of the PolyLacO-tagged rearranged Igλ locus. PolyLacO isintegrated between ΨV17-20; other notions as in FIG. 4A. FIG. 5B: Cellcycle profile of DT40 and DT40 PolyLacO-λR cells. FIG. 5C: Accumulationof sIgM-loss variants by DT40 and DT40 PolyLacO-λR cells. Frequencies ofsIgM-loss variants in 24 subclones from each line were quantitated byflow cytometry following 6 wk clonal expansion. Frequencies shown werenormalized to DT40; mean sIgM-loss frequencies were 0.8% and 0.9%,respectively. FIG. 5D: Cell cycle profile of DT40 PolyLacO-λR GFP-LacIand DT40 PolyLacO-λR E47-LacI cells. FIG. 5E: RT-PCR analysis ofexpression of Igλ, AID or β-actin mRNAs in DT40 PolyLacO-λR GFP-LacI andDT40 PolyLacO-λR E47-LacI transfectants. Triangles indicate 30, 10, 3and 1× relative concentrations of cDNA templates. FIG. 5F: Mean sIgMloss of independent clonal DT40 PolyLacO-λR GFP-LacI (n=13) and DT40PolyLacO-λR E47-LacI (n=19) transfectants, cultured for 3 wk in theabsence or presence of 100 μM IPTG. Values were normalized to DT40PolyLacO-λR GFP-LacI cells cultured without IPTG.

FIG. 6A-B. Nuclear radius correlates with cell cycle. FIG. 6A:Representative images of G1- and G2/M-enriched cells. G1 (left) and G2/M(right) cells were stained with Hoechst 33342 (10 μM; Molecular Probes)then sorted based on DNA content. Bar, 10 μm. FIG. 6B: Representativecell cycle profile of DT40 PolyLacO-λR cells. Mean radii±s.d. are shownas horizontal bars within the representative profile. Dotted verticallines indicate cut-offs for G1 and G2 used in experimental analyses: G1,r<4 μm; G2, r>5.2 μm.

FIG. 7A-D. E2A localizes to λR in G1 phase of cell cycle. FIG. 7A:Representative image of colocalization of λR and E2A in DT40 PolyLacO-λRGFP-LacI cells. Nuclear perimeter as determined by DAPI staining isoutlined by the dashed white line. Bar, 5 μm. FIG. 7B: Fraction ofλR/E2A colocalizations occurring in each phase of cell cycle in DT40PolyLacO-λR

GFP-LacI cells. FIG. 7C: Representative image of colocalization of λRand active Pol II (P*-Pol II) in DT40 PolyLacO-λR RFP-LacI cells.Notations as in 7A. FIG. 7D: Fraction of λR/P*-Pol II colocalizationsoccurring in each phase of cell cycle in DT40 PolyLacO-λR RFP-LacIcells.

FIG. 8A-F. Id1 expression inhibits λR/E2A colocalizations in G1 phase.FIG. 8A: Western blot assaying Id1 expression in DT40 PolyLacO-λRRFP-LacI (left) and DT40 PolyLacO-λR RFP-LacI Id1 cells (right). Markerpolypeptide sizes (kDa) at left. FIG. 8B: Cell cycle profile of DT40PolyLacO-λR RFP-LacI and DT40 PolyLacO-λR RFP-LacI Id1 cells. FIG. 8C:Mean slgM loss of independent clonal DT40 PolyLacO-λR RFP-LacI Id1transfectants (n=6), cultured for 6 wk. Values were normalized to DT40PolyLacO-λR RFP-LacI cells. FIG. 8D: Id1 expression does not alter Igλor AID transcript levels. RT-PCR analysis of expression of Igλ, AID orβ-actin control mRNA in DT40 PolyLacO-λR REP-Lad and DT40 PolyLacO-λRRFP-LacI Id1 cells. FIG. 8E: Effect of Id1 expression on cellcycle-dependence of λR/E2A colocalizations in DT40 PolyLacO-λR RFP-LacIcells and a derivative stably expressing Id1 (Id1 − and +,respectively). Percent of colocalizations in each stage of cell cycle isshown. FIG. 8F: Effect of Id1 expression on cell cycle-dependence ofcolocalizations of λR/P*-Pol II colocalizations and in DT40 PolyLacO-λRRFP-LacI cells and a derivative stably expressing Id1. Details as in 8E.

FIG. 9A-B. Chromatin Modification at the DT40 Igλ Locus. FIG. 9A:Schematic diagram of the rearranged chicken Igλ locus, showing the 25ΨVλ regions and the rearranged VλR gene (leader, L; variable region,Vλ-Jλ; constant region, Cλ region. FIG. 9B: Summary of a representativechromatin immunoprecipitation experiment, assaying N-terminalacetylation of histones H3 and H4 (AcH3 and AcH4). Sites interrogatedwere: a region approximately 1 kb upstream of the ΨVλ array (flank); theregion between ΨVλ5 and Vλ; ΨVλ1; ΨVλ5; ΨVλ13; ΨVλ18; ΨVλ24; ΨVλ25; andthe rearranged VλR and unrearranged Vλu alleles. See Materials andMethods for details. Bars indicate standard deviation.

FIG. 10A-B. Reversible Tethering of GFP-LacI to the ΨVλ Array in DT40PolyLacO-λR. FIG. 10A: Schematic diagram of the rearranged chicken Igλlocus in DT40, with polymerized lactose operator (PolyLacO) insertedbetween ΨVλ17-20. Notations as in FIG. 9. FIG. 10B: Fluorescent imagesof DT40 GFP-LacI transfectants and DT40 PolyLacO-VλR GFP-LacItransfectants cultured in the absence of IPTG (left); or in the presenceof 100 μM IPTG overnight (right).

FIG. 11A-C. Tethered HP1 Diminishes Modifications Characteristic ofActive Chromatin. FIG. 11A: Representative fluorescent images of singleDT40 PolyLacO-VλR LacI-HP1 transfectants, stained with anti-LacIantibodies (left); DAPI (center); or merged image (right). FIG. 11B:Enrichment of acetylated H3 (AcH3) and H4 (AcH4) at ΨVλ17R in DT40PolyLacO-VλR GFP-LacI and DT40 PolyLacO-VλR LacI-HP1 transfectants.Following ChIP, duplex PCR was carried out with Ova and ωVλ17R primers.Enrichment is expressed relative to the total DNA input control,±standard deviation of four separate amplifications of increasingamounts of template DNA. FIG. 11C: Histogram showing enrichment of AcH3and AcH4 at ΨVλ17R (from panel B) and the pol ε promoter in DT40PolyLacO-VλR GFP-LacI and DT40 PolyLacO-VλR LacI-HP1 transfectants. Barsindicate standard deviation.

FIG. 12A-C. Tethered HP1 Does Not Affect IgA Expression. FIG. 12A:Relative enrichment of AcH3 and AcH4 at VλR in DT40 PolyLacO-VλRGFP-LacI and DT40 PolyLacO-VλR LacI-HP1 transfectants. Enrichment valueswere normalized to the DT40 PolyLacO-VλR GFP-LacI control. Bars indicatestandard deviation of four separate amplifications of increasing amountsof template DNA. FIG. 12B: Relative intensity of cell surface IgM (sIgM)expression in DT40 PolyLacO-λR GFP-LacI (GFP-LacI) and DT40 PolyLacO-λRLacI-HP1 (LacI-HP1) transfectants cultured in the presence and absenceof 250 μM IPTG. sIgM levels were quantitated by measuring intensity ofstaining with mouse anti-chicken IgM antibody, and normalized to thelevel in DT40 PolyLacO-λR GFP-LacI transfectants. Details as in 12A.FIG. 12C: Relative levels of VλR transcripts in DT40 PolyLacO-λRGFP-LacI (GFP-LacI) and DT40 PolyLacO-λR LacI-HP1 (LacI-HP1)transfectants. Transcript levels were quantitated by RT-PCR andnormalized to the level in DT40 PolyLacO-λR GFP-LacI transfectants.Details as in 12A.

FIG. 13A-B. Tethered HP1 Decreases Histone Acetylation throughout theΨVλ Array. FIG. 13A: Summary of a chromatin immunoprecipitationexperiment, assaying N-terminal acetylation of histone H3 in chromatinfrom the DT40 PolyLacO-VλR LacI-HP1 cell line cultured for 3 days in thepresence and absence of 250 μM IPTG. ChIP enrichment values (see Example4) were normalized to values obtained from a parallel analysis ofchromatin from DT40 PolyLacO-VλR GFP-LacI cells. Bars indicate standarddeviation of four separate amplifications of increasing amounts oftemplate DNA. FIG. 13B: Summary of a chromatin immunoprecipitationexperiment, assaying N-terminal acetylation of histones H4 in chromatinfrom the DT40 PolyLacO-VλR LacI-HP1 cell line cultured for 3 days in thepresence and absence of 250 μM IPTG. Details as in 13A.

FIG. 14A-E. Nontemplated and Templated Mutation Promoted by TetheredHP1. FIG. 14A: sIgM loss fluctuation assay of panels of independent DT40PolyLacO-VλR GFP-LacI (n=27) and DT40 PolyLacO-VλR LacI-HP1 (n=16). Thefigure shows combined data from at least two independent transfectantsfor each fusion construct. Median diversification rates are shown below.FIG. 14B: Summary of sequence analysis of Vλ regions carrying uniquemutations, from DT40 PolyLacO-VλR GFP-LacI (n=78) and DT40 PolyLacO-VλRLacI-HP1 (n=36) transfectants, as analyzed by single cell PCR. Sequenceswere pooled from two independent transfectants.

FIG. 15. Sequence Alignment of Mutated DT40 PolyLacO-λR LacI-HP1 Clones.Sequences of 14 unique, mutated VX regions from diversified DT40PolyLacO-λR LacI-HP1 cells. Blue boxes outline gene conversion tracts;red circles denote point mutations; black dotted boxes indicatenontemplated insertions; orange triangles denote deletions. Parentsequence of DT40 region (SEQ ID NO: 31) is presented, with mutationsites indicated.

FIG. 16A-E. Ig gene conversion is accelerated by VP16 tethered to theΨVλ array. FIG. 16A: Schematic diagram of the rearranged chicken IgAlocus in DT40, with polymerized lactose operator (PolyLacO) insertedbetween ΨVλ17-20. Leader, L; rearranged variable region, Vλ-Jλ; constantregion, Cλ. FIG. 16B: Western blot analysis of expression of GFP-LacI orGFP-LacI-VP16 in indicated transfectants. Expression was assayed byblotting with anti-LacI antibodies, relative to β-actin control. FIG.16C: Cell cycle profile of DT40 PolyLacO-λ GFP-LacI and DT40 PolyLacO-λGFP-LacI-VP16 transfectants. FIG. 16D: Quantitation of RT-PCR comparisonof Vλ transcript levels in DT40 PolyLacO-λ GFP-LacI and DT40 PolyLacO-λGFP-LacI-VP16 transfectant, normalized to DT40 PolyLacO-λ GFP-LacI. FIG.16E: Fluorescent image of DT40 PolyLacO-λ GFP-LacI-VP16 transfectants.Arrows indicate GFP-LacI-VP16 bound to Igλ. Nuclei were counterstainedwith DAPI (blue).

FIG. 17A-C. AcH3 and AcH4 levels are increased and Ig gene conversion isaccelerated by VP16 tethered to the ΨVλ, array. FIG. 17A: Enrichment ofacetylated H3 (AcH3) at Igλ in DT40 PolyLacO-λ GFP-LacI-VP16transfectants. Following ChIP, duplex PCR was carried out with Ova andΨVλ17R primers. Enrichment is expressed relative to the total DNA inputcontrol, ±standard deviation of four separate amplifications ofincreasing amounts of template DNA. FIG. 17B: Enrichment of acetylatedH4 (AcH4) at Igλ in DT40 PolyLacO-λ GFP-LacI-VP16 transfectants.Notations as in 17A. FIG. 17C: Median slgM loss in panels of independentDT40 PolyLacO-λ GFP-LacI-VP16 transfectants, normalized to DT40PolyLacO-Vλ GFP-LacI transfectants. The figure shows combined data fromat least two independent transfectants for each fusion construct. Thefold increase relative to control DT40 PolyLacO-Vλ GFP-LacItransfectants is shown below.

FIG. 18A-B. The H3.3 histone variant is enriched at the DT40 Igλ locus.FIG. 18A: Schematic diagram of the rearranged chicken Igλ locus in DT40.Notations as in FIG. 16A. FIG. 18B: Summary of a representativechromatin immunoprecipitation experiment, assaying the H3.3 histonevariant. Sites interrogated were: a region approximately 1 kb upstreamof the ΨVλ array (flank); the region between ΨVλ5 and Vλ; ΨVλ1; ΨVλ5;ΨVλ13; ΨVλ18; ΨVλ24; ΨVλ25; and the rearranged VλR allele. Bars indicatestandard deviation.

FIG. 19A-E. Ig gene conversion is accelerated by HIRA tethered to theΨVλ array. FIG. 19A: Western blot analysis of expression of GFP-LacI orHIRA-LacI in indicated transfectants. Expression was assayed by blottingwith anti-LacI antibodies, relative to β-actin control. FIG. 19B: Cellcycle profile of DT40 PolyLacO-λ GFP-LacI and DT40 PolyLacO-λ HIRA-LacItransfectants. FIG. 19C: Quantitation of RT-PCR comparison of VXtranscript levels in DT40 PolyLacO-λ GFP-LacI and DT40 PolyLacO-λHIRA-LacI transfectants, normalized to DT40 PolyLacO-λ GFP-LacI. FIG.19E: Fluorescent image of DT40 PolyLacO-λ GFP-LacI-VP16 transfectants.Arrows indicate GFP-LacI-VP16 bound to Igλ. Nuclei were counterstainedwith DAPI (blue).

FIG. 20A-C. The H3.3 histone variant is enriched and gene conversionaccelerated by HIRA tethering in DT40 PolyLacO-λ. FIG. 20A: Enrichmentof H3.3 (green) and pan H3 (blue) at ΨVλ17R in DT40 PolyLacO-λ, and DT40PolyLacO-λ HIRA-LacI cells expressing H3.3-FLAG. Results for twoindependent lines of DT40 PolyLacO-λ HIRA-LacI are shown. Enrichment ofDT40 PolyLacO-λ is normalized to a value of “1”. Bars are shown±standarddeviation of four separate amplifications of increasing amounts oftemplate DNA. FIG. 20B: Flow cytometry of sIgM+ cells in representativepopulations of DT40 PolyLacO-GFP-LacI cells and DT40 PolyLacO-λHIRA-LacIcells. FIG. 20C: Median slgM loss in panels of independent DT40PolyLacO-λHIRA-LacI, and DT40 HIRA transfectants, normalized to DT40PolyLacO-λ GFP-LacI transfectants. The figure shows combined data fromat least two independent transfectants for each fusion construct. Thefold increase relative to control DT40 PolyLacO-λ GFP-LacI transfectantsis shown below.

FIG. 21A-B. Distinct chromatin effects of tethered HIRA and VP16. FIG.21A: Southern blot analysis of the PolyLacO region in DT40 PolyLacO-λGFP-LacI, DT40 PolyLacO-λ GFP-LacI-VP16 and DT40 PolyLacO-λ HIRA-LacItransfectants, following digestion of chromatin with micrococcalnuclease (MNase). No MNase, (−). Numbers at the right denote nucleosomemultimers. FIG. 20B: Model of how elevated histone deposition could beresponsible for an accentuated “laddering” pattern of chromatin.Arrowheads indicate possible MNase cleavage sites. Dark arrowheadsindicate a restricted range of cleavable DNA, and grey arrowheadssuggest a large range of cleavable DNA to generate deviations from thepredicted ˜150 by cleavage product.

FIG. 22. Sequences of mutated V regions from single DT40 PolyLacO-λGFP-LacI-VP16 cells. V regions were amplified from single sIgM− cellsand then sequenced. Clear blue boxes outline long-tract gene conversionevents; blue-shaded boxes outline short-tract gene conversion events;red circles denote point mutations; black dotted boxes indicateinsertions; carats denote deletions.

FIG. 23. Sequences of mutated V regions from single DT40 PolyLacO-λHIRA-LacI cells. Notations as in FIG. 22.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the development of B cell lines inwhich mutagenesis of a target immunoglobulin gene is inducible.Immunoglobulin is expressed on the cell surface, permitting selectionfor clones that express immunoglobulin molecules with particularspecificities, increased affinities, and/or new activities. This can becarried out with immunoglobulin and also non-immunoglobulin genes at thetarget site.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “polypeptide” includes proteins, fragments of proteins,and peptides, whether isolated from natural sources, produced byrecombinant techniques or chemically synthesized. Peptides of theinvention typically comprise at least about 6 amino acids.

As used herein, “diversification” of a target gene means a change ormutation in sequence or structure of the target gene. Diversificationincludes the biological processes of somatic hypermutation, geneconversion, and class switch recombination, which can result in pointmutation, templated mutation DNA deletion and DNA insertion. Thediversification factors of the invention can induce, enhance or regulateany of these methods of diversification.

A “mutation” is an alteration of a polynucleotide sequence,characterized either by an alteration in one or more nucleotide bases,or by an insertion of one or more nucleotides into the sequence, or by adeletion of one or more nucleotides from the sequence, or a combinationof these.

As used herein, a “cis-regulatory element” is a DNA sequence positionedin a region that controls expression or diversification of a gene. Atethering factor binds in a sequence-specific manner to this region ofthe DNA. Representative cis-regulatory elements include, but are notlimited to, LacO and TetO.

As used herein, a “tethering factor” is a molecule that binds to thecis-regulatory element in a sequence-specific manner. One example of atethering factor is a “repressor”, a protein that is synthesized by aregulator gene and binds to an operator locus, blocking transcription ofthat operon. Exemplary tethering factors include, but are not limitedto, LacI and TetR.

As used herein, a “diversification factor” refers to a molecule thataccelerates or regulates diversification or hypermutation.Representative diversification factors include some identified initiallyas transcriptional regulators (e.g., VP16 or E47), aheterochromatin-associated protein (e.g., HP1), a histone chaperone(HIRA), a chromatin remodeler, a component of the nuclear pore complex(NUP153), a gene regulator or a combination thereof. Other moleculesthat can serve as a diversification factor include, but are not limitedto, a DNA repair factor, a DNA replication factor, a resolvase, ahelicase, a cell cycle regulator, a ubquitylation factor, a sumoylationfactor, or a combination thereof.

As used herein, “promoter” means a region of DNA, generally upstream(5′) of a coding region, which controls at least in part the initiationand level of transcription. Reference herein to a “promoter” is to betaken in its broadest context and includes the transcriptionalregulatory sequences of a classical genomic gene, including a TATA boxor a non-TATA box promoter, as well as additional regulatory elements(i.e., activating sequences, enhancers and silencers) that alter geneexpression in response to developmental and/or environmental stimuli, orin a tissue-specific or cell-type-specific manner. A promoter isusually, but not necessarily, positioned upstream or 5′, of a structuralgene, the expression of which it regulates. Furthermore, the regulatoryelements comprising a promoter are usually positioned within 2 kb of thestart site of transcription of the gene, although they may also be manykb away. Promoters may contain additional specific regulatory elements,located more distal to the start site to further enhance expression in acell, and/or to alter the timing or inducibility of expression of astructural gene to which it is operably connected.

As used herein, “operably connected” or “operably linked” and the likeis meant a linkage of polynucleotide elements in a functionalrelationship. A nucleic acid is “operably linked” when it is placed intoa functional relationship with another nucleic acid sequence. Forinstance, a promoter or enhancer is operably linked to a coding sequenceif it affects the transcription of the coding sequence. Operably linkedmeans that the nucleic acid sequences being linked are typicallycontiguous and, where necessary to join two protein coding regions,contiguous and in reading frame. “Operably linking” a promoter to atranscribable polynucleotide is meant placing the transcribablepolynucleotide (e.g., protein encoding polynucleotide or othertranscript) under the regulatory control of a promoter, which thencontrols the transcription and optionally translation of thatpolynucleotide.

The term “nucleic acid” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogs of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally-occurring nucleotides.

As used herein, “a” or “an” means at least one, unless clearly indicatedotherwise.

B Cells

The invention provides a B cell modified to permit reversible inductionof diversification of a target gene. The cell comprises a cis-regulatoryelement operably linked to a target gene of interest. A factor thatmodulates diversification can then be fused to a tethering factor thatbinds to the cis-regulatory element, thereby tethering thediversification factor to the region that controls expression of thetarget gene. The B cell can be a chicken DT40 B cell or other vertebrateB cell, with a human B cell or a chicken DT40 B cell containinghumanized immunoglobulin (Ig) genes (in which human IgH and IgL replacechicken IgH and IgL) preferred for some embodiments.

B cells are natural producers of antibodies, making them an attractivecell for production of both improved antibodies and improvednon-immunoglobulin proteins and polypeptides. DT40 B cells are aneffective starting point for evolving specific and high affinityantibodies by iterative cycles of hypermutation and selection (Cumberset al., 2002; Seo et al., 2005). DT40 cells have several advantages overother vehicles tested for this purpose. DT40 constitutively diversifiesits Ig genes in culture, and targets mutations to the CDRs of theexpressed antibody molecules. DT40 proliferates more rapidly than humanB cell lines (10-12 hr generation time, compared to 24 hr); clonalpopulations can be readily isolated because cells are easily cloned bylimiting dilution, without addition of special factors or feeder layers;and DT40 carries out efficient homologous gene targeting (Sale, 2004),so specific loci can be replaced at will allowing one to manipulatefactors that regulate hypermutation.

The invention provides a novel platform for generating high affinityantibodies. In one embodiment, the vehicle for antibody evolution is a Bcell line, DT40, which naturally produces antibodies, and which has beenengineered to make immunoglobulin gene hypermutation accelerated andinducible. Like other B cells, DT40 expresses antibodies on the cellsurface, allowing convenient clonal selection for high affinity andoptimized specificity, by fluorescence or magnetic-activated cellsorting. In the DT40 cell line, hypermutation is carried out by the samepathway that has been perfected over millions of years of vertebrateevolution to Ig gene hypermutation in a physiological context. Thishighly conserved pathway targets mutations preferentially (though notexclusively) to the complementarity-determining regions (CDRs), thesubdomains of the variable (V) regions that make contact with antigen(FIG. 1).

Thus far, the use of DT40 (and other cultured B cell lines) for antibodyselection has been limited because the rate of hypermutation is veryslow, about 0.1%-1% that of physiological hypermutation. To acceleratehypermutation, key regulatory sites and factors have been manipulated,taking advantage of our current sophisticated understanding of themolecular mechanisms of hypermutation.

Although chicken DT40 B cells offer many advantages, in some embodimentsit may be desired to use human B cells. Alternatively, one can employhumanized Ig genes with the chicken DT40 B cells. By humanizing the DT40immunoglobulin genes, the utility of this platform for therapeutics canbe broadened, as the antibodies generated in the DT40 platform could beused directly for treatment.

There is ample documentation of the utility of humanized antibody genes,and a number of validated approaches for humanization, as reviewedrecently (Waldmann and Morris, 2006; Almagro and Fransson, 2008).Humanization is effected by substitution of human Ig genes for thechicken Ig genes, and this is readily done in DT40 by taking advantageof the high efficiency of homologous gene targeting. The substitutionsare carried out by procedures analogous to those described in theexamples below to insert cis-regulatory elements, but using differenttargeting constructs that are designed to modify distinct parts of theheavy and light chain loci. Substitution could produce DT40 derivativesthat generate entirely humanized antibodies, by swapping V(D)J and Cregions; or chimeric antibodies (humanized C regions but not V regions).These replacements will not alter the adjacent cis-regulatory elementsor affect their ability to accelerate hypermutation. The conservedmechanisms that promote hypermutation will target mutagenesis to theCDRs of humanized sequences. The humanized line can thus be used foraccelerated development of human monoclonals in cell culture, providinga dual platform for rapid production of useful antibodies for eithertherapeutic or diagnostic purposes.

In addition, one can optimize antibody effector function by C regionreplacement. Antibody-based immunotherapy is a powerful approach fortherapy, but this approach thus far been limited in part by availabilityof specific antibodies with useful effector properties (Hung et al.,2008; Liu et al., 2008). The constant (C) region of an antibodydetermines effector function. Substitutions of either native orengineered human C regions can be made by homologous gene targeting inthe DT40 vehicle to generate antibodies with desired effector function.

Target Genes

Typically, the target gene comprises a promoter and a coding region. Thecoding region of the target gene in the B cell of the invention can beone that encodes any protein or peptide of interest, and need notcomprise a complete coding region. In some embodiments, a particularregion or domain is targeted for diversification, and the coding regionmay optionally encode only a portion that includes the region or domainof interest.

In one embodiment, the target gene comprises an immunoglobulin (Ig)gene, wherein the Ig gene comprises an Ig gene enhancer and codingregion. The Ig gene can be all or part of an IgL and/or IgH gene. Thecoding region can be native to the Ig gene, or a heterologous gene. Insome embodiments, the target gene is or contains a non-Ig target domainfor diversification, as well as domains permitting display of the geneproduct on the B cell surface, including a transmembrane domain and acytoplasmic tail.

Cis-Regulatory Element

The cis-regulatory element provides a landing pad in the region thatcontrols expression of the target gene. This landing pad provides aplace to which a tethering factor can bind in a sequence-specific mannerto this region of the DNA. A variety of molecules can be used ascis-regulatory elements, so long as the element serves the landing padfunction of providing a place to which a tethering factor (asequence-specific DNA binding protein) can bind to the DNA and bring adiversification factor, fused to the tethering factor, into sufficientproximity of the coding region so that diversification of the codingregion is capable of reversible regulation. In a typical embodiment, thecis-regulatory element is a polymerized Lactose operator (LacO). In oneembodiment, the element comprises about 80-100 repeats of LacO. Inanother embodiment, the cis-regulatory element is a tetracyclineoperator (TetO).

Tethering & Diversification Factors

A tethering factor is one that binds to the cis-regulatory element in asequence-specific manner. In some embodiments, regulation ofdiversification is achieved by using a tethering factor characterized byregulatable binding to the cis-regulatory element. In the embodiments inwhich LacO serves as a cis-regulatory element, the Lac repressor, LacI,can serve as the tethering factor, and its binding to the cis-regulatoryelement, LacO, can be regulated by isopropyl-β-D-thio-galactoside(IPTG). In the absence of IPTG, LacI binds LacO and diversification isaccelerated (or otherwise regulated) by the presence of thediversification factor. IPTG can be added in the event that a halt orreduction in activity of the diversification factor is desired, therebymaking the mutagenesis process a reversible one. In embodiments in whichTetO serves as the cis-regulatory element, TetR can be a suitabletethering factor, and the activity of the diversification factor can beregulated by tetracycline or doxycycline.

A diversification factor is one that enables modulation of mutagenesisin the B cell. The examples below describe the effects of tethering andreleasing several regulatory factors. One, the heterochromatin proteinHP1, accelerated hypermutation 5.6-fold (Example 4); another, E47-LacI(E47 is one isoform of the E2A regulator, critical to B celldevelopment), accelerated hypermutation 4.5-fold (Example 3); and twoother modifiers of chromatin structure accelerated hypermutation8.4-fold and 11.0-fold (FIG. 2). In addition, a tethering site has beentargeted to the expressed Ig heavy chain locus. Dual regulation ofhypermutation at IgH and lgL can accelerate hypermutation from 25- to100-fold, enabling rapid production of new antibodies.

Diversifying immunoglobulin genes move within the nucleus in the courseof cell cycle, and this movement is correlated with and necessary fordiversification. Tethering to the nuclear pore by expression ofNP153-LacI in DT40 PolyLacO-λ cells (using methods described in theexamples below) accelerates diversification 5.7-fold. This resultpredicts that other factors that regulate gene position also regulatediversification, including other nuclear pore proteins and other factorsthat determine or regulate gene position in the nucleus.

Chromatin structure regulates diversification as well. The data inExample 4 show that expression of the heterochromatin protein HP1 fusedto LacI accelerates diversification and that this effect is reversibleby IPTG. This finding has been extended to the histone chaperone, HIRA.

Regulators of gene expression or function also serve as diversificationfactors. Example 3 below shows that expression of the regulatory factorE2A (expressed as the E47 isoform, as a LacI fusion) acceleratesdiversification, and that this effect is reversible by IPTG. The VP16regulatory domain from herpes virus accelerates diversification, andthat this effect is reversible by IPTG.

Deamination also accelerates mutagenesis, but it is not inducible. AIDis the B cell-specific DNA deaminase that initiates Ig genediversification. Example 2 below shows that tethering AID (AID-LacI) inDT40 PolyLacO-λ promotes mutagenesis, but that IPTG does not overcomethis effect. Thus, it appears that mutagenic affects are genome-wide,making this factor unsuitable without further modification.

In some embodiments, the diversification factor is a transcriptionalregulator, a heterochromatin-associated protein, a histone chaperone, achromatin remodeler, a component of the nuclear pore complex, a generegulator or a combination thereof. Other molecules that can serve as adiversification factor include, but are not limited to, a DNA repairfactor, a DNA replication factor, a resolvase, a helicase, a cell cycleregulator, a ubquitylation factor, a sumoylation factor, or acombination thereof. In one embodiment, the transcriptional regulator isVP16 or E47. A typical heterochromatin-associated protein for use as adiversification factor is HP1. A representative histone chaperone isHIRA.

TABLE 1 Representative Diversification Factors Transcriptionalactivators VP16 NeuroD TFIIB A13, B1, E47 NPAS TFIID B2, B3, E12 OLIGTFIIE B4, B5, B6, Myc TAL1 TFIIF B7, c-Fos Twist TFIIH B8, B9, B13,c-Jun USF1 GFI1 C4, BACH MAX HIC C5, C6, C8, BATF MITF HIVEF C9, BLZF1MNT IKZF C10, C11, C/EBP MLX ILF C13, D1, D3, CREB MXI1 KLF D4, D8, D9,CREM SREBP MTF1 C10, D11, DBP AP-2 MYT1 D12, D13) DDIT3 CAR OSR1 HOPXGABPA FXR Sp1 MEIS HLF LXR Zbtb7 MEOX2 MAF PPAR ZB1 MNX1 NFE PXR HIVEP1MSX NRL RAR AIRE NANOG NRF1 ROR DIDO1 NKX XBP1 Rev-ErbA GRLF1 PHF ATOH1HNF4 ING POU domain AhR PNR JARID proteins AHRR RXR JMJD1B OTX ARNT NURARX PDX ASCL1 GCNF CDX PAX BHLHB2 DAX1 CRX E2F (1-5) BMAL GATA CUTL1 FOXCLOCK ATBF1 DLX proteins EPAS1 CTCF EMX2 HSF HAND E4F1 EN ELF HES EGRFHL EGF HEY ERV3 ESX1 ELK HIF ATBF1 HHEX ERF ID BCL HLX ERG LYL1 CTCFHomeobox ETS MXD4 E4F1 (A1, A3, A4, ETV MYCL1 EGR A5, FLI1 MYCN ERV3 A7,A9, A10, MYB MyoD TFIIA A11, MYBL2 NFAT SRY RBL2 NF-κB STAT SSRP1Apetala 2 NFI Mef2 CSDA EREBP NFY SRF YBX1 B3 Rho/Sigma HNF (1A, CBFARID R-SMAD 1B) HMGA CAP LEF1 HBP1 IFI SOX Rb MLL RBL1 MNDAHeterochromatin- Chromatin associated proteins Histone chaperonesremodelers HP1 a Polycomb proteins Nucleolin HP1b Trithorax proteinsRtt106 HP1c HIRA NAP1 ASF1a NAP2 ASFb CAF1 Spt6 DNA repair XRCC2 HAP1CSB BRG1 DMC1 APE1 XPV BAF RAD52 DNA EndoV PBAF RecA polymerase MSH2 BRMRecB b MSH3 Rdh54 RecC DNA Ligase MSH5 XRCC3 RecD I MSH6 FANCD2 MRE11XRCC1 MLH1 FANCI RAD50 PARP PMS2 CtIP XRS2/NBS1 XPA EXO1 Rad54 KU70 XPBPOLH Rad51 KU80 XPC POLI BRCA1 SAE2 XPD REV1 BRCA2 DNA-PK XPE POLQRAD51B DNA Ligase XPF RAD51C iV XPG RAD51D UNG CSA Resolvases and Cellcycle DNA Replication Helicases regulators PCNA RuvC CHK1 RPA RusA CHK2Smc1 RuvB p53 Smc3 RuvC p27 Rad21/Mcd1/Scc1 RecQ Stromalin/Scc3 hRECQ5Scc2 WRN Scc4 BLM Pds5 Srs2 Eco1 hRECQ1/L Rec8 RTS/hRECQ4 Gene HASPINMLL3 SET1A Regulation MLL1 MLL4 SET1B CARM1 MLL2 MLL5 SET7/9 ASH1JMD2A/JHDM NSD1 Sc ESA1 LSD/BHC110 3A SMYD2 SIRT2 PRMT5 JMJD2B SET2 SIR2SUV39H1 JMJD2C/GAS JHDM1a SUV4-20H1 SUV39H2 C1 JHDM1b SUV4-20H2SETDB1/ESE JMJ2D2D Sc RTT109 PR-SET7/8 T JHDM2a DOT1 SET9 EuHMTase/GLJHDM2b CKII Bmi/Ring1A P AuroraB PRMT4 MST1 G9a CBP/p300 PRMT5RNF20/RNF40 CLL8 EZH2 HBO1 UbcH6 RIZ1 MSK1 Sc HAT1 PCAF MSK2 TIP60 GCN5FPR4 HOB1 Nuclear pore complex Nup153 Nup98 CRM1 other stable anddynamic pore components Ubiquitylation IAP Sumoylation E1 Cdc34 Ubc9 E2Rbx1 SAE1/Aos1 E3 NEDD8/Rub1 SAE2/Uba2 26S proteasome SCF/Grr1 SUMOE2 E6SCF/Met30 PIAS VHL SCF/Fbw7/hCdc4 Ulp1 MDM2 Cue1p Siz1 BAP1 Ubc7p Siz2BARD1 Ubc1p SUMO E1 CBL CHIP SUMO E3 SCF/Skp2 Hsp/c70/90 Nse2 (Mms21)Skp1 Parkin Ulp2 Cul1/Cdc53 NEDD4-2 DUB Csn5

Fusion Constructs

The tethering of a diversification factor to the cis-regulatory elementis achieved via fusion of the diversification factor to a tetheringfactor. Fusion constructs encoding this combination of factors can bepresent in and expressed by the B cell of the invention, and can also beprovided separately, to be added to the B cell at the desired time andupon selection of the desired factors for a particular objective.

Fusion constructs may generally be prepared using standard techniques.For example, DNA sequences encoding the peptide components may beassembled separately, and ligated into an appropriate expression vector.The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The 3′ end of theDNA sequence encoding one peptide component is ligated, with or withouta peptide linker, to the 5′ end of a DNA sequence encoding the secondpeptide component so that the reading frames of the sequences are inphase. This permits translation into a single fusion protein thatretains the biological activity of both component peptides.

A peptide linker sequence may be employed to separate the first and thesecond peptide components by a distance sufficient to ensure that eachpeptide folds into its secondary and tertiary structures. Such a peptidelinker sequence is incorporated into the fusion protein using standardtechniques well known in the art. Suitable peptide linker sequences maybe chosen based on the following factors: (1) their ability to adopt aflexible extended conformation; (2) their inability to adopt a secondarystructure that could interact with functional regions on the first andsecond peptides; and (3) the lack of hydrophobic or charged residuesthat might react with the peptide functional regions. Preferred peptidelinker sequences contain Gly, Asn and Ser residues. Other near neutralamino acids, such as Thr and Ala may also be used in the linkersequence.

Methods and Uses of the Invention

The invention provides a method of producing a repertoire ofpolypeptides having variant sequences of a polypeptide of interest viadiversification of polynucleotide sequences that encode the polypeptide.Typically, the method comprises culturing the B cell of the invention inconditions that allow expression of the diversification factor, whereinthe target gene of the B cell contains the coding region of thepolypeptide of interest, thereby permitting diversification of thecoding region. The method can further comprise maintaining the cultureunder conditions that permit proliferation of the B cell until aplurality of variant polypeptides and the desired repertoire isobtained. Because the B cells express the polypeptides on the externalsurface, the nature and extent of the repertoire can be determined. Therepertoire can then be used for selection of polypeptides having desiredproperties.

In another embodiment, the invention provides a method of producing Bcells that produce an optimized polypeptide of interest. The methodcomprises culturing a B cell of the invention in conditions that allowexpression of the diversification factor, wherein the target gene of theB cell contains the coding region of the polypeptide of interest, andwherein the B cell expresses the polypeptide of interest on its surface.The method further comprises selecting cells from the culture thatexpress the polypeptide of interest on the B cell surface by selectingcells that bind a ligand that specifically binds the polypeptide ofinterest. These steps of culturing and selecting can be repeated untilcells are selected that have a desired affinity for the ligand thatspecifically binds the polypeptide of interest. The evolution ofantibody specificities during expansion of a B cell clone is illustratedin FIG. 3. Selection of a population that initially expresses multiplespecificities (top left) successively enriches for a desired specificity(center) and higher affinity (bottom right).

In embodiments in which the polypeptide of interest is an Ig, such as anIgL, IgH or both, the ligand may be a polypeptide, produced byrecombinant or other means, that represents an antigen. The ligand canbe bound to or linked to a solid support to facilitate selection, forexample, by magnetic-activated cell selection (MACS). In anotherexample, the ligand can be bound to or linked to a fluorescent tag, toallow for or fluorescence-activated cell sorting (FACS). Those skilledin the art appreciate that other methods of labeling and selecting cellsare known and can be used in this method.

The methods of the invention can further comprise adding a regulatorymolecule to the culture, wherein the regulatory molecule modulatesbinding of the tethering factor to the cis-regulatory element, therebymodulating diversification of the coding region. In the examplesdiscussed above, IPTG, tetracycline and doxycycline serve as theregulatory molecules. Those skilled in the art are aware of otherregulatory molecules that can be used with a particular tethering factorto regulate diversification activity.

The regulatory molecule can modulate the diversification of the codingregion in a variety of ways. For example, in some embodiments, theregulatory molecule is added to the culture to effect modulation, andthe modulation can result in reducing or halting diversification, or inenhancing or accelerating diversification, depending on whether theparticular regulatory molecule is one that increases or decreasesbinding of the tethering factor to the cis-regulatory element, and onwhether that particular change in binding has the effect of increasingor decreasing diversification activity. In other embodiments, themodulation, be it reduction, halt, enhancement or acceleration of themodulation, is effected by removing or eliminating the regulatorymolecule from the culture. Likewise, modulation of diversification canbe effected by adding a gene to or eliminating a gene from the B cell,or by increasing or diminishing the expression of a gene in the B cell.One skilled in the art can readily appreciate all of the availablepermutations set forth above, each of which has the effect of alteringthe level or presence of a regulatory molecule in the B cell, and inturn, altering the tethering of the diversification factor to thecis-regulatory element and thereby altering the diversificationactivity.

Additional uses of the methods of the invention include induciblehypermutation of Ig gene targets at Igλ. The DT40 PolyLacO-λ. LacI-HP1cell line (see Example 4 below) can be used for altering the sequence ofany Ig gene downstream of the Vλ pseudo-V array. The genomic structureat the Ig loci has evolved to promote mutagenesis of the V region butnot the constant region of an Ig molecule. Altering Ig gene sequence inthe context of an Ig locus takes advantage of that to ensure that theproduct of mutagenesis retains a functional constant region. The ease ofgene replacement in DT40 permits useful changes in that gene. Forexample, the chicken gene (variable and constant region) could bereplaced with a human antibody gene (either heavy or light chain), togenerate antibodies with therapeutic application. This can also be usedto generate therapeutic antibodies for other species. This will providea rapid system for mutagenesis of either only one chain of aheterodimeric antibody; or for the single chain of single chainantibodies.

Uses of this approach include: hypermutation to generate B cell clonesthat produce high affinity Igs; hypermutation to alter cross-reactivityof antibodies, while retaining recognition of a specific epitope;hypermutation to identify V region sequences with high affinity forspecific compounds. This can be done at only a single Ig locus, or both.

Inducible hypermutation can also be performed at IgH. The IgH locus inDT40 is an efficient site of mutagenesis, just like Igλ. Derivative celllines, made by parallel techniques, permit mutagenesis at both IgH andIgλ, or at each allele separately. Insertion of PolyLacO at IgH, tocreate the DT40 PolyLacO-λ PolyLacO-H LacI-HP1 derivative, permitshypermutation at both H and L chain genes. Adding IPTG to the culturemedium releases some of the inhibitory effects that diminish templatedevents and increase nontemplated hypermutation. Thus, by varying thepresence and absence of IPTG, one can toggle back and forth betweentemplated and non-templated mutagenesis of the genes that encode bothchains of an antibody.

A derivative can be made that combines regulation with IPTG of LacRepressor (LacI) at one allele with tetracycline of Tet repressor (TetR)regulation at the other allele. This TetR derivative will permitindependent regulation of diversification at the IgH and IgL alleles;and using different mechanisms simultaneously to regulatediversification at the IgL and IgH locus.

The invention also provides a vehicle for selection of T cell receptors.T cell-based immunotherapy has great potential (Blattman and Greenberg,2004). T cell receptor specificity and affinity is governed by CDRcontacts (Chlewicki et al., 2005). Accelerated inducible selection forspecificity or high affinity T cell receptors can be carried out in aDT40 PolyLacO vehicle, which has been modified by substitution of T cellreceptors (V regions or entire genes) for the Ig loci.

Production of catalytic Igs is another aspect of the invention. TheIg-related methods of the invention are not simply limited to theproduction of Igs for binding and recognition, as the target Ig couldalso be used for catalysis. After development of a stable molecule thatmimics the transition state of an enzymatic reaction, DT40 PolyLacO-λLacI-HP1 cells can be used to evolve an antibody that binds andstabilizes the actual chemical transition state. After identifyingclones that produce an Ig capable of binding the intermediate, thesystem can be used again to screen for catalytic activity of Igs on thereal substrate in culture. Once some activity has been demonstrated inthis system, optimization of activity can proceed by further evolutionof the Ig loci through mutagenesis. Thus, invention does not requireanimal immunization (a slow step), immortalization by hybridomatechnology, and the inefficiency of later having to screen hybridomasfor antibodies that demonstrate catalytic activity.

Inducible hypermutation of non-Ig gene targets at Igλ or IgH is anotheraspect of the invention. The genomic structure at the Ig loci hasevolved to promote mutagenesis of 1-1.5 kb downstream of the promoter.This system can be harnessed to mutate short regions of genes. Clonalselection based on surface protein expression can be incorporated byfusion of the region of interest to a portion of a gene expressingelements that mediate surface expression. Exemplary elements for surfaceexpression include a signal peptide, transmembrane domain andcytoplasmic tail from a protein expressed on the B cell surface (Chou etal., 1999; Liao et al., 2001).

The invention can also be used for the production of recognition arrays.The ability to evolve cells harboring receptors with affinities for alarge spectrum of antigens allows the development of recognition arrays.Combining this technology with intracellular responses/signaling fromreceptor stimulation in DT40 (such as measurement of Ca2+ by aequorin(Rider et al., 2003) or use of reporter gene transcription) would createa useful biosensor. Diversified clones would be spotted into arrays or96 well plates, and exposed to samples. Each sample would yield a“fingerprint” of stimulation. The arrays would permit qualitativecomparisons of biological/medical, environmental, and chemical samples.Analysis need not be limited to the analysis of proteins, as is the casefor comparative techniques like 2D gels, since all forms of compoundscould have antigenic properties. Furthermore, the arrays would lead tothe identification of components without knowledge of their presencebeforehand.

Kits

For use in the methods described herein, kits are also within the scopeof the invention. Such kits can comprise a carrier, package or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements (e.g., cells, constructs) to be used in themethod.

Typically, the kit comprises a B cell of the invention and fusionconstructs that express the corresponding tethering and diversificationfactors. For example, the B cell comprises a cis-regulatory elementoperably linked to a target gene, wherein the target gene comprises apromoter and a coding region. The kit further comprises one or morecontainers, with one or more fusion constructs stored in the containers.Each fusion construct comprises a polynucleotide that can be expressedin the B cell and that encodes a tethering factor fused to adiversification factor, wherein the tethering factor specifically bindsto the cis-regulatory element of the B cell. The B cell can include aplurality of cis-regulatory elements for use with a plurality of fusionconstructs.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. In addition, a label can be provided on the container to indicatethat the composition is used for a specific therapeutic ornon-therapeutic application, and can also indicate directions for use.Directions and or other information can also be included on an insertwhich is included with the kit.

EXAMPLES

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1 Mutation and Targeting in the DT40 B Cell Line

This example illustrates an embodiment of the invention, prepared in twosteps.

(1) DT40 PolyLacO-λ. We first constructed this derivative of the chickenB cell line DT40 containing a genetic element which allows regulation incis at the Igλ locus. In DT40 PolyLacO-λ, a polymerized Lactose operator(LacO) is inserted just upstream of the pseudo-VX array at the Igλ lightchain locus. This is an extension of a system developed by Straight andBelmont, in which PolyLacO consists of approximately 80 repeats of the20 by LacO binding site (Straight et al., 1996; Belmont, 2001). We haveshown that this does not affect the normal process of templatedmutagenesis. Construction of DT40PolyLacO-λ is described in detail inExample 2 below.

LacO is bound by Lactose repressor (Lad) with very high affinity: <10⁻¹²M. This mechanism regulates expression of the operon for metabolism oflactose in E. coli, and can also be used in a variety of cellularcontexts. We have shown that Lac! binds to PolyLacO in DT40 PolyLacO-λ,by generating stable transfectants that express LacI fused tofluorescent protein (GFP, RFP, YFP or CFP), and showing byimmunofluorescence microscopy that bound protein can be imaged as asingle spot in the nucleus of normally proliferating DT40 PolyLac stablyexpressing GFP-LacI, RFP-LacI, YFP-LacI or CFP-LacI.

IPTG is a small molecule which causes release of LacI from LacO, both invitro and in cultured cells. Culture of DT40 PolyLacO-λ GFP-LacI with atlittle as 10 μM IPTG causes release of GFP-LacI from PolyLacO. Culturewith IPTG does not affect cell proliferation.

(2) DT40 PolyLacO-λ LacI-HP1. We next constructed this derivative ofDT40 PolyLacO-λ, in which a switch to somatic hypermutation occurs whenDrosophila melanogaster HP1 (from L. Wallrath, University of Iowa), amodifier of chromatin structure, is tethered to PolyLacO. Tethering wasachieved using the very well-characterized pair of cis-andtrans-regulators, E. coli LacO/LacI. HP1 is a well-characterizednon-histone heterochromatin protein that functions in heterochromaticgene silencing and the spreading of heterochromatin, and in Drosophilatethered LacI-HP1 has been shown to promote a closed chromatin structureand inactivation of reporter genes neighboring a lacO repeat (Li et al.,2003; Danzer and Wallrath, 2004). We demonstrated that tethering HP1caused a transition of the donor sequences from an open to nonpermissivestate, as measured by ChIP; and caused VX to undergo mutagenesis bysomatic hypermutation rather than gene conversion. This is functionallyequivalent to deletion of the pseudo-VX array, which has been shown tocause a switch from templated mutagenesis to somatic hypermutation(Arakawa et al., 2004). Further characterization of DT40 PolyLacO-λ.LacI-HP1 is documented in Example 4 below.

(3) DT40 PolyLacO-λ VP16-LacI. We next used the expression plasmidp3′ss-EGFP-VP16 (from A. Belmont; University of Illinois) to tetherVP16-LacI to PolyLacO. VP16 is a strong activator of transcription, andit has been demonstrated to recruit histone acetyltransferase complexes(Tumbar et al., 1999). We demonstrated that tethering VP16 created amore permissive chromatin structure as measured by ChIP; and caused Vλto undergo mutagenesis by a stimulation of gene conversion overwild-type levels.

Example 2 Acceleration of Mutagenesis by Tethering Genes to the NuclearPore

We discovered that diversifying immunoglobulin genes move within thenucleus in the course of the cell cycle, that this movement iscorrelated with the steps in the diversification pathway, and thatdiversification initiates at the nuclear periphery. Activation InducedDeaminase (AID), the enzyme that initiates diversification, carries anuclear export signal and is constantly transported out of the nucleusvia the nuclear pore, so its concentrations may be higher at theperiphery of the nucleus. This suggested that it might be possible toaccelerate diversification by increasing gene proximity to the nuclearpore. We showed that this is the case by using a fusion of the nuclearpore protein, Nup153, to Lac repressor (Lad) to tether the IgH locus inDT40PolyLacO to the nuclear pore. We found that this accelerated theclonal diversification rate 5.7-fold. Control experiments showed thattethering to PolyLac° was critical to acceleration of mutagenesis. Thismethod is a general one that depends on the AID nuclear export signaland gene localization to promote mutagenesis of a target. Thus thismethod can be extended to promote mutagenesis of non-Ig genes in Bcells, by tethering them to the pore; and further extended to promotemutagenesis of genes in non-B cells, by expressing AID in those cells.

Example 3 E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig GeneDiversification

This example describes cell cycle-dependent regulation of Ig genediversification in the nucleus. Rearranged Ig genes undergodiversification in sequence and structure initiated by the DNAdeaminase, AID. Ig genes must be transcribed for diversification tooccur, but whether there are additional requirements for cis-activationhas not been established. This example shows, by chromatinimmunoprecipitation, that the regulatory factor E2A associates with therearranged IgλR gene in the chicken DT40 B cell line, which carries outconstitutive Ig gene diversification. By direct imaging of a DT40derivative in which polymerized lactose operator tags the rearranged λRgene, we show that λR/E2A colocalizations are most prominent in G1 phaseof cell cycle. We further show that expression of the E2A antagonist Id1prevents λR/E2A colocalizations in G1 phase, and impairs diversificationbut not transcription of λR (hereinbelow, the IgλR gene is sometimesreferred to as Igλ or λ). Thus, E2A acts in cis to promote Ig genediversification, and G1 phase is the critical window for E2A action.

The regulated changes in genomic sequence and structure that take placeat the Ig loci reflect both targeting of DNA damage to these genes, andescape from faithful repair. Somatic hypermutation, class switchrecombination and gene conversion are all initiated by the Bcell-specific enzyme, activation-induced deaminase (AID) (5-8). AIDdeaminates cytosine to uracil, with clear preference for single-strandedDNA (9-11). Transcription is prerequisite for diversification, which mayreflect preference of AID for single-stranded substrates. Uracil in DNAis a common lesion, which can be repaired faithfully by highly conservedand efficient pathways (12). However, the Ig loci can escape fromfaithful repair and undergo repair by error-prone pathways (13).

E2A, a member of the E family of bHLH proteins, is a critical regulatorof many aspects of lymphocyte development (14-17). E proteins dimerizeto bind to the E box motif, CANNTG, and their function is antagonized byId proteins, which heterodimerize with E proteins to prevent DNAbinding. E2A is induced in activated murine B cells, where it regulatesclass switch recombination (18) as well as expression of the gene thatencodes AID (19). In chicken B cells, inactivation of the E2A geneimpairs IgA gene diversification but not transcription (20, 21); while,conversely, ectopic expression of E47 (one of two functionallyequivalent isoforms encoded by E2A) promotes Igλ gene diversification,but does not affect IgA transcript levels (22).

The possibility that E2A might regulate Ig gene diversification bybinding to sites in cis was first suggested by evidence thatmultimerized E-boxes stimulate hypermutation but not transcription of anIg transgene in mice (23). This possibility has been further supportedby the demonstration that multimerized E-boxes can promote Ig genediversification but not transcription in chicken B cells (24). However,clear resolution of the question of whether E2A acts directly at the Iggenes to promote diversification has been difficult, for severalreasons. E-boxes function as sites for E2A-dependent regulation only inspecific contexts, so the presence of an E-box does not guarantee E2Afunction at a site; the loose consensus and frequent occurrence of E-boxmotifs precludes mutational analysis of each individual site; and atsome loci E2A is recruited by protein-protein rather than protein-DNAinteraction, so an E-box is not always prerequisite for E2A-dependentregulation (25).

We have now established that E2A acts in cis at the Ig genes to promotediversification, in experiments which take advantage of derivatives ofthe constitutively diversifying chicken B cell line, DT40, in which therearranged Igλ allele is tagged with polymerized lactose operator (DT40PolyLacO-λR). By chromatin immunoprecipitation (ChIP), we show that E2Aassociates with the rearranged but not unrearranged IgA allele in theparental line, DT40. This example demonstrates that, in DT40 PolyLacO-λRcells, diversification is accelerated upon expression of an E47-LacIfusion protein, which effectively tethers E47 to λR; and that thestimulatory effect of E47-LacI expression is not evident in cellscultured with IPTG, so binding in cis is necessary to promotediversification. By direct imaging of the rearranged λR gene in DT40PolyLacO-λR GFP-LacI cells, we show that λR/E2A colocalizationspredominate in G1 phase; and that expression of the E2A antagonist, Id1,impairs diversification and diminishes λR/E2A colocalizationsspecifically in G1 phase, but does not affect A transcript levels orlocalization of λR to active transcription factories. These results showthat E2A acts in cis in G1 phase to promote Ig gene diversification.

Materials and Methods

Cell Culture, Transfection, sIgM Loss Assay and Cell Cycle Analysis

The chicken bursal lymphoma line DT40 and its derivative DT40PolyLacO-λ_(R) were maintained and transfected as described (26, 27).The E47-LacI expression construct was generated by subcloning of E47cDNA from the S003 E47 plasmid (28) (provided by Cornelis Murre,University of California, San Diego, Calif.) into the p3′SS-GFP-LacIplasmid (provided by Andrew Belmont, University of Illinois, Urbana,Ill.). The Id1 expression construct (29) was provided by Barbara Christy(University of Texas, San Antonio, Tex.). The sIgM-loss assay wascarried out as described (26, 30), and results compared using theMann-Whitney U test with the R software package(http://www.r-project.org). For cell-cycle profiles based on DNAcontent, 1×10⁶ exponentially growing cells were suspended in 0.1% TritonX-100, treated with 200 μg/ml RNase A and 50 μg/ml propidium iodide, andanalyzed as described (26).

ChIP Analysis

Chromatin was prepared and immunoprecipitated as described (27, 31, 32);using anti-E2A antibody (ab11176; Abcam) or control IgG.Semiquantitative PCR was performed with FastStart Taq DNA polymerase(Roche), using previously described primers for Vλ_(R) and Vλ_(U) (27);and primers 5′-ATTGCGCATTGTTATCCACA-3′ (SEQ ID NO: 1) and5′-TAAGCCCTGCCAGTTCTCAT-3′ (SEQ ID NO: 2) for ovalbumin (Ova). PCRproducts were quantitated with ImageQuant software (Amersham).Enrichment was calculated as the ratio of the amplicon of interest tothe Ova amplicon, normalized to the ratio from control IgG, e.g.:Enrichment Vλ_(R)=(anti-E2A [Vλ_(R)/Ova])/(IgG [Vλ_(R)/Ova]).

RT-PCR and Western Blotting

For RT-PCR assays, AID and b-actin were amplified with primers asdescribed (7); and Igλ transcripts with primers5′-GTCAGCAAACCCAGGAGAAAC-3′ (SEQ ID NO: 3) and5′-AATCCACAGTCACTGGGCTG-3′ (SEQ ID NO: 4). For Western blotting, wholecell lysates (50 μg) from DT40 PolyLacO-λ_(R) RFP-LacI and DT40PolyLacO-λ_(R) RFP-LacI Id1 were resolved, and Id1 protein was detectedwith anti-Id1 antibody (JC-FL; Santa Cruz) using FluorChem HD2 (AlphaInnotech).

Fluorescence Microscopy and Image Analysis

To image PolyLacO, DT40 PolyLacO-I_(R) cells were transfected with theGFP-LacI expression construct, p3′SS-GFP-LacI (from Andrew Belmont,University of Illinois, Urbana, Ill.), which encodes LacI engineered tocontain SV40 nuclear localization signals and lacking a sequencenecessary for tetramer formation (33); or its derivative, RFP-LacI, inwhich GFP was replaced with RFP (DsRed-monomer; Clontech). Forimmunostaining, cells (˜3×10⁵) were deposited onto glass slides usingCytospin3 (800 rpm, 4 min; Shandon), fixed with 2% paraformaldehyde for20 min, and stained as described previously (26). Primary antibodiesused were: anti-E2A (ab11176, 1:200; Abcam); anti-Pol II C-terminaldomain phosphorylated at Ser5 (ab5131, 1:500; Abcam). Alexa Fluor 488-or 594-conjugated anti-IgG (Molecular Probes) was used as secondaryantibodies. Fluorescent images were acquired using the DeltaVisionmicroscopy system (Applied Precision) and processed and analyzed withsoftWoRx (Applied Precision) and Imaris softwares (Bitplane).Fluorescent signals were sometimes partially rather than completelyoverlapping, which may reflect the considerable distance (˜17 kb)between the PolyLacO-tag and the VX region; both configurations werescored as colocalization. Fraction of colocalization was analyzed withPearson's χ² test. Nuclear radii were calculated as the average of atleast two independent measurements of diameter, divided by two. Cellcycle dependence of mean nuclear radius was determined independently foreach cell line, and proved to be relatively invariable. Standard valuesused to correlate nuclear radius to cell cycle were: G1, r<4 μm; G2,r≧5.2 μm.

Results E2A Associates With the Rearranged But Not Unrearranged Igλ Gene

Despite the considerable evidence for the importance of E2A in Ig genediversification, this factor had not been shown to associate directlywith the Ig genes. To test association of E2A with Igλ, we used anti-E2Aantibodies to immunoprecipitate chromatin from the chicken DT40 B cellline. This line was derived from a bursal lymphoma and carries outconstitutive diversification of both Ig heavy and light chain genes bygene conversion. A search of the 11 kb chicken λ light chain locusidentified more than 50 matches to the E2A consensus, CANNTG: 17 in theregion between Vλ and ΨVλ1, the most proximal of the upstreampseudogenes; 2 in the matrix attachment region (MAR) in the J-C intron;and 6 in the 3′ enhancer. In DT40 B cells, the functional allele hasundergone VJ recombination early in B cell development, which deletes a1.8-kb region to join the V and J segments, while the inactive A alleleis unrearranged (FIG. 4A), allowing the two alleles to be readilydistinguished by PCR. Following chromatin immunoprecipitation (ChIP),recovery of the rearranged and unrearranged λ alleles was assayedrelative to a control gene, ovalbumin. This showed that E2A was17.6-fold enriched at the rearranged VλR allele, but not enriched at theunrearranged VλU allele (FIG. 4B). Thus E2A associates directly with therearranged VλR allele.

E2A Acts in cis to Regulate Igλ Diversification

To ask if E2A must bind in cis to promote diversification, we tookadvantage of a derivative of DT40, DT40 PolyLacO-λR, in whichpolymerized lactose operator has been inserted in the ΨVλ array byhomologous gene targeting (FIG. 5A), allowing factors expressed asfusions with lactose repressor (Lac!) to be tethered to the rearrangedλR allele and released by culture with IPTG (27). Cell cycledistribution and clonal rates of Ig gene conversion were comparable inDT40 PolyLacO-λR and wild-type DT40 (FIG. 5B, C). DT40 PolyLacO-λR cellswere stably transfected with a plasmid expressing the E47 isoform of E2Afused to LacI (E47-LacI), or a control plasmid expressing greenfluorescent protein fused to LacI (GFP-LacI). Cell cycle distributionwas comparable in GFP-LacI and E47-LacI transfectants, although culturesof the latter line contained some sub-G1 (apoptotic) cells (FIG. 5D).Levels of Igλ transcripts were unaltered in the E47-LacI transfectants(FIG. 5E), confirming published results showing that E2A does notregulate Ig gene expression in chicken B cells (21, 22). Levels of AIDtranscripts were approximately 3-fold higher in the E47-LacItransfectants (FIG. 5E). A similar increase in AID expression inresponse to ectopic expression of E47 has been observed by others (22).

To ask if E2A regulates diversification directly, via binding to IgA, wecultured independent E47-LacI (n=19) or GFP-LacI (n=13) transfectants inthe presence and absence of IPTG, and determined clonal diversificationrates using the slgM loss fluctuation assay (26, 27, 30). This assayscores inactivating mutations regardless of whether they occur by geneconversion, point mutation, deletion or insertion, and thus quantitatesinitiating events independent of the outcome of mutagenesis. Thisanalysis showed that the clonal rate of diversification was 4.5-foldhigher in E47-LacI transfectants relative to GFP-LacI controls (P=0.019, Mann-Whitney U test; FIG. 5F). Moreover, culture with IPTG,which releases E47-LacI from PolyLacO, caused diversification rates toreturn almost to background levels in DT40 PolyLacO-λR E47-LacI cells,but had no effect on GFP-LacI controls (FIG. 5F). Thus E47-LacI promotesdiversification by acting in cis.

E2A Localizes to IgλR in G1 Phase of Cell Cycle

In human B cells, receptor crosslinking in G1 phase of cell cycle caninitiate somatic hypermutation, producing identifiable mutations within90 minutes (34). Thus it was of interest to determine the stage of cellcycle in which E2A acts at Igλ. The rearranged and diversifying λR genecan readily be imaged as a bright dot in DT40 PolyLacO-λR cellsexpressing GFP-LacI (27). However, when cells were stained with Hoechst33342 and sorted by DNA content to enrich for cells in G1, S, or G2/Mstage, the fraction of cells exhibiting a clear fluorescent signal fromthe tagged gene diminished from the 90-95% routinely observed inunsorted cells to approximately 45%. As such a loss in signal could biasresults, we therefore determined cell cycle stage by a differentapproach. Analysis of Hoechst 33342-stained and sorted cells showed thatnuclear size was significantly smaller in G1 phase than in G2/M phasecells (e.g. FIG. 6A). We therefore asked if nuclear radius (r) could beused to establish the stage of cell cycle, by measuring nuclear radii ofG1 cells (n=55) and G2 cells (n=55) from an exponentially growing DT40PolyLacO-λR population which had been stained with Hoechst 33342 andsorted based on DNA content. Mean nuclear radii of G1 cells was 3.8±0.3μm; and of G2 cells, 5.4±0.5 μm (FIG. 6B). Comparison of the ratios ofG1:S:G2 cells as determined by nuclear radius (3:6:1) and staining(2.7:5.5:1.7) further validated this approach. Thus, G1 cells wereidentified experimentally as r<4 μm; and G2 cells, r>5.2 μm.

Colocalizations of λR/E2A were readily identified by deconvolutionmicroscopic analysis of DT40 PolyLacO-λR GFP-LacI cells stained withanti-E2A antibodies (e.g. FIG. 7A). λR/E2A colocalizations were evidentin 26% of asynchronous cells (n=227). Analysis of the cell cycledistribution of colocalizations showed that 45% of λR/E2Acolocalizations occurred in G1 phase, 38% in S phase, and 17% in G2phase cells (FIG. 7B). Thus, there was an apparent excess of λR/E2Acolocalizations in G1 phase (45%) relative to the fraction (25%) of G1phase cells (P<0.0001, χ2 test).

We also determined the cell cycle-dependence of λR transcription,identifying active transcription factories by staining with antibody tophosphorylated Ser5 in the C-terminal domain of RNA polymerase II(P*-Pol II), a modification characteristic of elongating Pol IImolecules (35). In asynchronous cell populations of DT40 PolyLacO-λRRFP-LacI cells, numerous active transcription factories could beidentified throughout the nucleus, and λR/P*-Pol II colocalizations werereadily observed in 19% of cells (n=392; e.g. FIG. 7C). Analysis of thecell cycle distribution of λR/P*-Pol II colocalizations showed that 21%of colocalizations occurred in G1 phase; 58% in S phase; and 21% in G2phase cells (FIG. 7D). This is comparable to the cell cycledistribution. Thus, λR is transcribed throughout the cell cycle, butλR/E2A colocalizations predominate in G1 phase.

Id1 Expression Inhibits Ig Gene Diversification and λR/E2AColocalizations in G1 Phase

To ask if λR/E2A colocalizations in G1 phase are critical todiversification, we determined the effect of Id expression on thesecolocalizations. Id antagonizes E2A, and expression in DT40 B cells ofId1 or Id3 has previously been shown to diminish Ig gene diversification(22). We generated stable DT40 PolyLacO-λR RFP-LacI Id1 transfectants,confirmed Id1 expression by Western blotting (FIG. 8A), and showed thatId1 expression did not alter the cell cycle profile (FIG. 8B). Weverified that Id1 expression diminished the clonal rate of Ig genediversification (P<0.001, Mann-Whitney U test; FIG. 8C); but did notaffect levels of Igλ or AID transcripts (FIG. 8D). We then comparedλR/E2A colocalizations in the Id1-expressing derivative and parentalline, by staining with anti-E2A antibodies. λR/E2A colocalizations wereevident in 13% of asynchronous DT40 PolyLacO-λR RFP-LacI Id1 cells(n=90), compared to 26% of DT40 PolyLacO-λR RFP-LacI cells (P=0.0030, χ2test).

To determine whether Id1 expression affected colocalizations in aspecific stage of cell cycle, λR/E2A colocalizations in the Id1transfectants were quantified with respect to nuclear radius and cellcycle. This showed that, in DT40 PolyLacO-λR RFP-LacI Id1 cells, 10% ofcolocalizations occurred in G1 phase; 58% in S phase; and 32% in G2phase cells (FIG. 8E). Thus, Id1 expression caused a significantdecrease in the fraction of λR/E2A colocalizations in G1 phase, from 45%in the parental line to 10% in Id1 transfectants (P<0.0001, χ2 test;FIG. 8E); and 90% of λR/E2A colocalizations in Id1 transfectantsoccurred after G1 phase of cell cycle. Taken together with diminisheddiversification evident in Id1 transfectants, this shows that G1 phaseis the critical window in which E2A promotes diversification.

λR/P*-Pol II colocalizations were identified in 18% of DT40 PolyLacO-λRRFP-LacI Id1 cells stained with antibodies to active transcriptionfactories (n=290), comparable to the parental line (19%, P=0.80, χ2test; FIG. 7D). The cell cycle profile of λR/P*-Pol II colocalizationswas also comparable in Id1 transfectants and the parental line (FIG.8F). The absence of effect of Id1 expression on λR/P*-Pol IIcolocalizations is consistent with undiminished Igλ transcript levels inDT40 PolyLacO-λR RFP-LacI Id1 transfectants (FIG. 8D). Thus, Id1expression affects the cell cycle distribution of colocalizations of λRwith E2A, but not with P*-Pol II.

Discussion

These results show that E2A must act in cis to promote Ig genediversification, and that G1 phase is the critical window in which E2Afunctions in this process. The experiments have examined A genes taggedwith PolyLacO and imaged by binding to GFP-LacI or RFP-LacI. Thisprovides a powerful approach for studying gene diversification. Thetagged locus is visible in >90% of fixed cells, enabling analysis ofcolocalizations with factors involved in diversification.

The ability to tether potential regulators and release by culture withIPTG makes it possible to study the effects of a factor at the Igλ locusindependent of its other targets. This is especially useful for a factorlike E2A, which functions at the top of a large and complex regulatoryhierarchy (36).

The results establish that E2A directly regulates Ig genediversification by physical association with the Ig loci. ChIP providedclear evidence for association of E2A with the rearranged λR allele inDT40 B cells. That E2A must function in cis was established by showingthat the acceleration in diversification resulting from tethering E2A(E47-LacI fusion) to the Igλ allele in DT40 PolyLacO-λR cells was notevident in cells cultured with IPTG, which releases LacI from LacO.

E2A is best-known as a transcriptional regulator, but E2A function indiversification does not reflect transcriptional activation at Igλ, aslevels of Igλ transcripts were not altered by ectopic expression of E47.This confirms results of others who have examined the effects of E2A ondiversification in chicken and murine B cells (20, 22, 23). In addition,colocalizations of λR with E2A, which are most prominent in G1 phase,distinct from the cell cycle distribution of λR to active transcriptionfactories, which is comparable to cell cycle distribution, suggestingthat λR is transcribed throughout the cell cycle. The independence ofE2A function in diversification and transcription is further supportedby the contrasting effects of Id1 expression on colocalizations of therearranged λR with E2A and active transcription factories. Id1expression diminished diversification, and also diminishedcolocalizations of λR with E2A. In contrast, Id1 expression did notaffect the number or cell cycle distribution of localization of λR toactive transcription factories. That E2A is not required to recruit λRto transcription factories is consistent with the absence of effect ofId1 expression on Igλ transcription.

E2A does regulate AID expression, and this indirectly stimulates Ig genediversification. The AID gene was shown to be a target oftranscriptional regulation by E2A in murine B cells (19); and we andothers (22) have shown that ectopic expression of E2A increases AIDtranscript levels in DT40 chicken B cells. This may contribute to oraccount for the modest acceleration in diversification evident inIPTG-cultured DT40 PolyLacO-λR E47-LacI cells. While E2A ablation hasbeen reported not to diminish AID transcript levels in chicken B cells(21), factors redundant with E2A might ensure a minimum level of AIDexpression in the absence of E2A.

The results identify G1 phase as the critical window in which E2Afunctions at Igλ. We observed an excess of λR/E2A colocalizations in G1phase, relative to other stages of cell cycle; and showed that Id1expression specifically diminishes colocalizations in G1 phase, anddecreases the diversification rate. Id proteins heterodimerize with Eproteins to inhibit DNA binding (14). That colocalizations during G1were specifically affected by Id1 expression may be indicative ofdistinct modes of E2A association with Igλ during cell cycle. In S andG2 phases, E2A may be recruited via interactions with other proteins,rather than by direct binding to DNA.

Additional lines of evidence support the view that diversification isinitiated in G1 phase. Somatic hypermutation in the human BL2 cell linecan be induced by in vitro stimulation that takes place only during G1phase, and point mutations first become evident within 90 minutes ofstimulation, when cells are still in G1 (34). In murine B cellsactivated for class switch recombination, IgH colocalizations with NBS1or γ-H2AX, participants in the switch recombination pathway, areprominent in G1 phase (37); and DNA breaks at the S regions can bedetected in G1 phase (38). DNA breaks have also been identified in laterstages of cell cycle in hypermutating human B cell lines (39), but theseproved to be AID-independent (40).

E2A may function in G1 phase to prepare a locus for events that occurlater in cell cycle, or even during a subsequent cell cycle. E2A hasbeen recently implicated in maintenance of histone H4 acetylation (21),and it is possible that E2A functions to establish a local chromatinenvironment favorable to AID attack or effective diversification indaughter cells.

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Example 4 Chromatin Structure Regulates Gene Conversion

This example illustrates how chromatin structure contributes to the useof homologous sequences as donors for repair using the chicken B cellline DT40 as a model. In DT40, immunoglobulin genes undergo regulatedsequence diversification by gene conversion templated by pseudogenedonors. We have found that the VX pseudogene array is characterized byhistone modifications associated with active chromatin. We have directlydemonstrated the importance of chromatin structure for gene conversion,using a regulatable experimental system in which the heterochromatinprotein, HP1, expressed as a fusion to Lac repressor, is tethered topolymerized lactose operators integrated within the pseudo-Vλ donorarray. Tethered HP1 diminished histone acetylation within the pseudo-Vλarray, and altered the outcome of Vλ diversification, so thatnontemplated mutations rather than templated mutations predominated.Thus, chromatin structure regulates homology-directed repair. Theseresults suggest that histone modifications may contribute to maintaininggenomic stability by preventing recombination between repetitivesequences.

This example uses the following abbreviations: AcH3, acetylated histoneH3; AcH4, acetylated histone H4; AID, activation-induced deaminase; DSB,double-strand break; GFP, green fluorescent protein; Ig, immunoglobulin;MRN, MRE11/RAD50/NBS1; NHEJ, nonhomologous end-joining; V, variable;UNG, Uracil DNA glycosylase; AP, abasic; PolyLacO, polymerized lactoseoperator; ChIP, chromatin immunoprecipitation; Ova, ovalbumin; LOH, Lossof heterozygosity.

Materials and Methods

Chromatin immunoprecipitation (ChIP). ChIP was carried out as previouslydescribed [48,89]. For all experiments at least two chromatinpreparations from at least two independent stably-transfected lines wereanalyzed. Figures present one representative experiment in which resultsfrom analysis of four separate amplifications were used to calculate astandard deviation. Four separate amplifications of serial dilutions oftemplate DNA were carried out, to establish that the measured productintensities were within the linear range. Enrichment of the experimentalamplicon was normalized to enrichment of an internal control ampliconfrom the ovalbumin (Ova) gene, amplified in the same tube by duplex PCR;and enrichment upon ChIP with specific antibodies was normalized toparallel experiments in which ChIP was carried out with total input DNAcontrols. Inclusion of the Ova internal control amplicon enabled us tonormalize for IP efficiency, background carryover, and differences ingel loading. Enrichment=

[(ΨVλ/Ova)Ab]/[(ΨVλ/Ova)Input]. As an additional control, the ratio ofthe experimental and control amplicons in the total input control wascompared to a control ChIP with polyspecific IgG; in all cases,enrichment in input and IgG controls were essentially equal. Data arepresented for representative experiments; standard deviations werecalculated from four separate amplifications of serial dilutions oftemplate DNA.

Antibodies used were: anti-AcH3 (06-599), anti-AcH4 (06-866), anddimethylated H3(K4) (07-030) from Upstate (Lake Placid, N.Y.). PCRprimers for ChIP were:

Vλ_(R): (SEQ ID NOS: 5-28, respectively)5′-GCCGTCACTGATTGCCGTTTTCTCCCCTC-3′ and5′-CGAGACGAGGTCAGCGACTCACCTAGGAC-3′; region between ψVλ1 and Vλ:5′-CTGTGGCCTGTCAGTGCTTA-3′ and 5′-GCAGGGAACCACAAGAACAT-3′; ψVλ1:5′-GGGACTTGTGTCACCAGGAT-3′ and 5′-CGCAGTCACATGTGGAATATC-3′; ψVλ5:5′-GAGCCCCATTTTCTCTCCTC-3′ and 5′-GAGATGTGCAGCAACAAGGA-3′; ψVλ13:5′-CCCTCTCCCTATGCAGGTTC-3′ and 5′-CCCCTATCACCATACCAGGA-3′; ψVλ18:5′-CCATTTTCTCCCCTCTCTCC-3′ and 5′-TCACCCTACAGCTTCAGTGC-3′; ψVλ24:5′-CCATTTTCTCCCCTCTCTCC-3′ and 5′-CAGCCCATCACTCCCTCTTA-3′; ψVλ25:5′-TCTGTTGGTTTCAGCACAGC-3′ and 5′-GCAGTTCTGTGGGATGAGGT-3′; ψVλ upstreamflank: 5′-GGCTCCTGTAGCTGATCCTG-3′ and 5′-GTTCTTTGCTCTTCGGTTGC-3′; ψVλ17at the PolyLacO-targeted allele: 5′-TAGATAGGGATAACAGGGTAATAGC-3′ and5′-AGGGCTGTACCTCAGTTTCAC-3′; OVA: 5′-ATTGCGCATTGTTATCCACA-3′ and5′-TAAGCCCTGCCAGTTCTCAT-3′; polε: 5′-GGGCTGGCTCATCAACAT-3′ and5′-CTGGGTGGCCACATAGAAGT-3′.

Constructs, transfection and cell culture. The LacI-HP1 expressionplasmid was created by substituting LacI-HP1a from a construct providedby L. Walirath (University of Iowa, Iowa City) for AID in pAIDPuro (fromH. Arakawa; Munich, Germany), to position LacI-HP1 downstream of thechicken β-actin promoter. The GFP-LacI expression plasmid(p3′ss-EGFP-LacI) was provided by A. Belmont (University of Illinois,Urbana). Cell culture and transfection were carried out as previouslydescribed [47]. DT40 PolyLacO-λ_(R) was generated by homologous genetargeting, using a construct carrying approximately 3.8 kb ofpolymerized lactose operator (PolyLacO) flanked by arms designed fortargeting the region between ΨVλ17-ΨVλ20, 17 kb upstream of thetranscribed Vλ_(R). In brief, homologous integrants were identified byPCR, and the selectable marker deleted by Cre expression. The DT40bursal lymphoma derives from B cell in which only one Igλ allele isrearranged, and in which the two parental chromomes are distinguished bya polymorphism near ΨVλ17. This enabled us to determine whether therearranged or unrearranged allele had been targeted by PCR. Controlexperiments established that cell cycle distribution was comparable inDT40 PolyLacO-λ_(R), DT40 PolyLacO-λ_(R) GFP-LacI and DT40PolyLacO-λ_(R) LacI-HP1 cells; and that culture of cells with up to 500μM IPTG for 7 days did not affect proliferation rate or chromatinmodifications at ΨVλ17_(R) in DT40 PolyLacO-λ_(R) GFP-LacI controlcells. Oligonucleotides for Vλ sequence analysis have been described[47].

Fluorescence imaging. For fluorescence imaging, cells (2×10⁵) werecytospun onto glass slides and fixed with 2% paraformaldehyde for 20min, permeabilized with 0.1% NP-40 for 15 min, and stained as previouslydescribed [90]. Primary staining was with an anti-LacI monoclonalantibody (1:500 dilution; Upstate); and the secondary antibody wasdonkey anti-mouse IgG Alexa Fluor 594 (1:2000; Molecular Probes, Eugene,Oreg.). To visualize the nucleus, cells were stained with DAPI (Sigma,Saint Louis, Mo.). Fluorescent images were acquired using theDeltaVision microscopy system (Applied Precision) and processed withsoftWoRx software (Applied Precision).

RT-PCR. RNA was harvested from cells using TRIzol Reagent (Invitrogen)and purified with a PreAnalytiX column (Qiagen). VA transcripts wereamplified following dilution of the template (1:1300); and β-actin wasamplified from an undiluted sample. The primers for amplification of Vλwere 5′-GTCAGCAAACCCAGGAGAAAC-3′ (SEQ ID NO: 29) and5′-AATCCACAGTCACTGGGCTG-3′ (SEQ ID NO: 30). The primers foramplification of β-actin have been described [36].

Quantitation of slgM loss variants and sequence analysis. The slgM lossvariant assay, which measures the accumulated slgM-loss variantsresulting from frameshift or nonsense mutations in mutated V regions,was used to quantitate Ig V region diversification [47,50]. In brief,sIgM⁺ cells were isolated by flow cytometry followed by limitingdilution cloning, and expanded for 4 weeks. To quantitate the fractionof sIgM⁻ cells, approximately 1×10⁶ cells were stained with anti-chickenIgM-RPE (Southern Biotechnology Associates, Birmingham, Ala.), andanalyzed on a FACScan with CellQuest software (BD Biosciences).

Single-cell PCR and sequence analysis were performed as described [47].In brief, sIgM⁻ cells were sorted, aliquoted to single wells, VA regionsamplified and sequenced, and their sequences compared to the ΨVλ donorsto determine if mutations were templated or nontemplated. The criterionfor a templated mutation was that nine consecutive bases must be anexact match in donor and recipient. Sequences derived from twoindependently transfected lines. Only unique sequences were included forclassification of the mutations.

Results Permissive Chromatin Structure at Vλ and ΨVλ Donor Templates

In DT40 B cells, the functional Vλ gene at one Igλ allele is rearrangedand expressed, and the other is unrearranged and not expressed. Wecharacterized chromatin structure at the rearranged (Vλ_(R)) andunrearranged (Vλ_(U)) alleles and the ΨVλ array by chromatinimmunoprecipitation (ChIP). ChIP was carried out with antibodiesspecific for lysine acetylation at the N-termini of histones H3 and H4.Recovered DNA was amplified in duplex PCR reactions; recovery normalizedto an amplicon from the ovalbumin (Ova) gene, which is not expressed inB cells; and enrichment normalized to a total DNA input control (seeMaterials and Methods for details). The distinct genomic structure ofVλ_(R) and Vλ_(U) permit them to be distinguished by PCR with specificprimers. ChIP demonstrated considerable enrichment of acetylatedhistones H3 and H4 (AcH3 and AcH4) at the rearranged Vλ_(R) gene. In atypical experiment, AcH3 was enriched more than 80-fold at Vλ_(R), andAcH4 more than 30-fold (FIG. 9B). In contrast, at the unrearrangedVλ_(U) allele, the levels of AcH3 and AcH4 were much lower than atVλ_(R) (16-fold and 7-fold lower, respectively); and only a few foldenriched relative to input DNA.

Chromatin structure within the ΨVλ array was assayed by amplificationwith primers which interrogated seven sites, including a region betweenΨVλ1 and the Vλ gene, ΨVλ1, ΨVλ5, ΨVλ13, ΨVλ18, ΨVλ24, ΨVλ25, and theupstream flanking region. (Due to a paucity of polymorphisms, the ΨVλarrays at the two Igλ alleles in DT40 cannot be readily distinguished byPCR.) Strikingly, we observed considerable enrichment of AcH3 and AcH4throughout the ΨVλ array (FIG. 9B). Enrichment was not proportional todistance from the transcribed Vλ_(R) gene, as sites distant from Vλ_(R)did not consistently display lower levels of enrichment than proximalsites (FIG. 9B). Thus, enrichment of acetylated histones within the ΨVλarray does not simply represent a graded spreading of chromatinmodification from the transcribed Vλ_(R) gene to sites upstream. Thenon-uniform chromatin structure of the locus suggests the presence ofcis-elements that regulate chromatin structure at the ΨVλ array.

Reversible Tethering of LacI Fusion Proteins to the ΨVλ Array in DT40PolyLacO-λ_(R)

Local modification of chromatin structure can be achieved by tetheringregulators to DNA binding sites as appropriate fusion proteins. Thisstrategy has, for example, been used to show that the heterochromatinprotein HP1a, expressed as a fusion with lactose repressor (LacI-HP1),promotes a closed chromatin structure and inactivation of reporter genesneighboring a LacO repeat in Drosophila [61,62]; and to show thattethering of the vertebrate G9a histone methyltransferase to a GAL4binding site within V(D)J minigene reporter impairsnonhomologous-mediated recombination of that construct [63]. The cellline, DT40 PolyLacO-λ_(R), which is a DT40 derivative in whichpolymerized lactose operator (PolyLacO) has been inserted by homologousgene targeting between ΨVλ17-ΨVλ20, 17 kb upstream of the transcribedVλ_(R) (FIG. 10A). The PolyLacO insert is 3.8 kb in length and comprisedof approximately 100 copies of a 20-mer operator [64]. Using this cellline, it is possible to assay the effects of tethered regulatory factorson homologous recombination in a physiological process within anendogenous locus, avoiding the need for a transgene reporter. Controlexperiments have shown that the PolyLacO tag does not affect cellproliferation, cell cycle, or Ig gene diversification.

In DT40 PolyLacO-λ_(R) GFP-LacI, which stably expresses enhanced greenfluorescent protein fused to lactose repressor (GFP-LacI), the taggedλ_(R) allele can be directly imaged by fluorescence microscopy andappears as a distinct dot in each cell (FIG. 10B, left). Tethering isreversible, as bright dots are not evident following overnight culturewith 100 μM IPTG, which prevents LacI from binding to LacO (FIG. 10B,right).

Tethered HP1 Diminishes Modifications Characteristic of Active Chromatinat ΨVλ

To manipulate chromatin structure at the ΨVλ array, we generated stabletransfectants of DT40 PolyLacO-λ_(R) which express the Drosophilamelanogaster HP1 protein fused to LacI (LacI-HP1). HP1 is a non-histoneheterochromatin protein that functions in heterochromatic genesilencing, the spreading of heterochromatin and histone deacetylation[58-60]. Tethered HP1 has been shown to promote a closed chromatinstructure at adjacent genes [61,62, 66-68]. Staining DT40 PolyLacO-λ_(R)LacI-HP1 transfectants with anti-LacI antibodies showed that LacI-HP1colocalized with DAPI dense-regions corresponding to pericentricheterochromatin (FIG. 11A), behaving as a functional marker ofheterochromatin [65].

To ask if tethered LacI-HP1 altered chromatin structure, we assayedchromatin modifications at ΨVλ17. This is the only site in the ΨVλ arrayat which the rearranged and unrearranged alleles can be distinguished byuse of specific PCR primers, due to a sequence polymorphism createdduring construction of DT40 PolyLacO. Following ChIP, DNA was amplifiedwith PCR primers specific for the targeted rearranged allele(ΨVλ17_(R)). Enrichment of ΨVλ17_(R) was compared to the nonexpressedovalbumin gene (Ova) as an internal control; and normalized to theΨVλ17_(R):Ova enrichment ratio in total input DNA (see Material andMethods). AcH3 and AcH4 were enriched at ΨVλ17_(R) in DT40PolyLacO-λ_(R) GFP-LacI controls 2.2-fold and 5.9-fold, respectively(FIG. 11B, C). These levels of enrichment are comparable to thosedocumented in DT40 (FIG. 9B). (Note that analysis of modification at ΨVλin the survey of the parental DT40 line necessarily includes bothalleles, which may underestimate activating modifications at therearranged allele. In contrast, analysis of modifications at ΨVλ17_(R)interrogates only the active allele.) AcH3 and AcH4 were not enriched atΨVλ17_(R) in DT40 PolyLacO-λ_(R) LacI-HP1 transfectants (0.6 and1.0-fold, respectively; FIG. 11B, C), consistent with HP1-mediatedsilencing. HP1 can effect silencing by recruitment of a histonemethyltransferase which modifies lysine 9 of histone H3 [66-68], but mayalso promote silencing independently of this modification [61]. ChIPusing antibodies against either di- and tri-methylated H3 (K9) did notreveal clear enrichment of the H3 K9-Me modification (data not shown).Methylation of lysine 4 (diMeK4) of histone H3 is associated withtranscription and generally exhibits an overlapping distribution withacetylation [69,70]. Assays of diMeK4(H3) at ΨVλ17_(R) demonstrated thatthis modification was 18.9-fold enriched in DT40 PolyLacO-λ_(R) GFP-LacIcells, but at background levels in DT40 PolyLacO-λ_(R) LacI-H P1 cells(FIG. 11B, C).

HP1 promotes maintenance and spreading of heterochromatin [66]. Toverify that changes in chromatin structure promoted by tethered HP1 didnot spread throughout the chromosome, we examined another site near theλ light chain locus on chromosome 15, the gene encoding the catalyticsubunit of DNA polε. DNA polε is ubiquitously expressed and essentialfor chromosomal replication in eukaryotes [71], and it is encoded by agene mapping approximately 2.1 Mb from Igλ. We found no difference inenrichment of AcH3 at the polε promoter region in the DT40PolyLacO-λ_(R) LacI-HP1 transfectants relative to DT40 PolyLacO-λ_(R)GFP-LacI controls (polε/Ova enrichment 8.5-fold and 8.4-fold,respectively; FIG. 3C). Similarly, there was no difference in AcH4 atthe polε promoter in the DT40 PolyLacO-λ_(R) LacI-HP1 transfectantsrelative to DT40 PolyLacO-λ_(R) GFP-LacI controls (polε/Ova enrichment1.9-fold and 1.7-fold, respectively; FIG. 11C). Thus, tethering ofLacI-HP1 at ΨVλ caused local modifications in chromatin structure,diminishing the AcH3, AcH4 and diMeK4(H3) modifications characteristicof open chromatin at ΨVλ17_(R), and causing chromatin to adopt a lesspermissive state.

Tethered HP1 Does Not Affect Vλ Gene Expression

We asked how tethered HP1 affected AcH3 and AcH4 levels at the expressedVλ_(R) by comparing these modifications in DT40 PolyLacO-λ_(R) LacI-HP1cells and the DT40 PolyLacO-λ_(R) GFP-LacI control transfectants (FIG.12A). Tethered HP1 diminished AcH3 and AcH4 levels to approximately 40%and 20% of the control levels, respectively. To ask if this affectedgene expression, we assayed both surface IgM (slgM) expression and Vλtranscript levels. Staining cells with mouse anti-chicken IgM showedthat slgM expression was comparable in DT40 PolyLacO-λ_(R) GFP-LacI andDT40 PolyLacO-λ_(R) LacI-HP1 lines, cultured in either the presence orabsence of IPTG (FIG. 12B). Vλ transcript levels were assayed byquantitative RT-PCR of RNA harvested from DT40 PolyLacO-λ_(R) GFP-LacIand D140 PolyLacO-λ_(R) LacI-HP1 cells, and normalized to β-actin as acontrol (FIG. 12C). No significant difference was observed between VAtranscript levels in the two cell lines, demonstrating thattranscription is not affected by tethering of HP1 within the ΨVλ array.Thus tethered LacI-HP1 did not affect expression of the downstream Iggene, although it did diminish AcH3 and AcH4 levels at Vλ_(R). The veryhigh AcH3 and AcH4 levels characteristic of Vλ (FIG. 9B, FIG. 12A) aretherefore not essential to maintain high levels of gene expression.

Tethered HP1 Alters Local Chromatin Structure

To assess how extensive the chromatin effects of LacI-HP1 were, weexamined AcH3 and AcH4 levels throughout the Igλ light chain locus atthe same amplicons examined in FIG. 9, including one in the flank, sixin the ΨVλ array, as well as at the expressed Vλ. Levels of modificationwere determined by comparing ΨVλ17_(R):Ova ratios of immunoprecipitatedand input conditions, as in FIG. 11 B. AcH3 modifications at the sitessurveyed ranged from 24% to 63% of the levels at the same sites in thecontrols (FIG. 13A, dark bars); and the average level of H3 acetylationacross all of the sites was 38% of the PolyLacO-λ_(R) GFP-LacI control.Culture of DT40 PolyLacO-λ_(R) LacI-HP1 transfectants for three dayswith 250 μM IPTG increased acetylation of H3 at all eight sites surveyed(FIG. 13A, compare dark and light bars). The effects of IPTG culturewere somewhat variable, but at most sites IPTG culture restored levelsof AcH3 to at least 45% of the level in the DT40 PolyLacO-λ_(R) GFP-LacIcontrol cells; with an average of over 80%. Thus, the chromatinmodifications at ΨVλ17_(R) in DT40 PolyLacO-λ_(R) LacI-HP1 cellsresulted directly from tethered LacI-HP1, and were largely reversible.

H4 acetylation was surveyed at the same eight sites (FIG. 13B, darkbars). AcH4 modifications were found to range from 18% to 42% of controllevels; and the average level was 29% of the control cell line. Culturewith IPTG for three days increased acetylation of H4 at all eight sitessurveyed (FIG. 13B, compare dark and light bars), restoring levels of H4acetylation to at least 57% of the level in the DT40 PolyLacO-λ_(R)GFP-LacI control cells; with an average of over 80%. Moreover, IPTG canat least partially reverse the effects of LacI-HP1.

These results show that the observed chromatin modifications in the ΨVλarray are due to tethering of HP1. Moreover, the fact that thesemodifications are reversible shows that an active mechanism reverseshistone modifications imposed by tethering chromatin modificationfactors at ΨVλ.

Tethered HP1 Impairs Templated Mutagenesis.

The ability to manipulate chromatin structure at ΨVλ by tetheringLacI-HP1 (FIGS. 11-13) enabled us to directly ask whether and howchromatin structure influences Ig gene conversion. We used the slgM lossvariant assay to determine if tethered LacI-HP1 affected the clonal rateof sequence diversification of the rearranged Vλ_(R) gene. Thisfluctuation assay measures the fraction of variant cells which no longerexpress structurally intact sIgM, and thus scores mutation eventsresulting from either gene conversion or point mutagenesis [47,50].Independent clonal derivatives of DT40 PolyLacO-λ_(R) GFP-LacI and DT40PolyLacO-λ_(R) LacI-HP1 were established by limiting dilution cloning ofslgM cells, and the fraction of sIgM⁻ cells in each populationdetermined by flow cytometry of cells cultured for 4 weeks, and thenstained with anti-IgM antibody. The median slgM loss rate was 0.5% forDT40 PolyLacO-λ_(R) GFP-LacI cells and 2.8% for DT40 PolyLacO-λ_(R)LacI-HP1 cells (FIG. 14A). This corresponds to 5.6-fold acceleration ofclonal diversification rates in LacI-HP1 transfectants relative toGFP-LacI controls.

Ig gene diversification in chicken B cells occurs predominantly by geneconversion (templated mutation); but if gene conversion is impaired, forexample by the absence of essential factors, repair can create asignificant fraction of nontemplated mutations [50-55]. This istypically accompanied by an increase in the clonal diversification rate,because the ΨVλ templates for gene conversion are about 80% identical tothe rearranged gene, and a significant fraction of DNA lesions that arerepaired by gene conversion do not undergo any alteration of sequence;in contrast, repair by a mutagenic polymerase is more likely to alterDNA sequence. To determine how tethering of HP1 accelerateddiversification, we sorted single sIgM⁻ cells from the DT40PolyLacO-λ_(R) GFP-LacI and DT40 PolyLacO-λ_(R) LacI-HP1 transfectants,amplified expressed Vλ regions by single-cell PCR, and sequenced theseregions. Sequence changes were categorized as templated if they werewithin a tract containing two or more mutations and the tract was anexact match to at least 9 by of a donor ΨVλ sequence; and as ambiguousif they consisted of only a single base change, but did match at least 9by of a donor ΨVλ sequence. Nontemplated events, consisting of pointmutations, deletions, and insertions, were also scored. In the controlDT40 PolyLacO-λ_(R) GFP-LacI transfectants, 54 templated events and 2ambiguous events were documented among 70 unique mutations; thus mostevents (77%) were templated, and a small fraction of events (20%) werepoint mutations (FIG. 14B, left; FIG. 15). Strikingly, in DT40PolyLacO-λ_(R) LacI-HP1 cells, point mutations predominated (59%),accompanied by deletions (8%) and insertions (14%); while only 1 clearlytemplated event and 6 ambiguous events were documented among 36 uniquemutations (FIG. 14B, right; FIG. 15). Thus only 3% of mutations wereclearly templated, and even including the ambiguous class of potentiallytemplated mutations, templating could account for no more than 19% ofmutation. Statistical comparisons showed that the difference between thefraction of clearly templated mutations in DT40 PolyLacO-λ_(R) GFP-LacIcontrol cells and DT40 PolyLacO-λ_(R) LacI-HP1 transfectants (77%compared to 3%) was highly significant (P=7.1×10⁻⁷; Fisher's exacttest). The difference in the fraction of ambiguous, potentiallytemplated mutations in the control cells (3%) and HP1 transfectants(17%) is also significant (P=0.05; Fisher's exact test). This suggeststhat some mutations in this category may arise as a result oflimitations on the length of a gene conversion tract imposed bynonpermissive donor chromatin. Thus, tethering of HP1 accelerated clonalrates of mutagenesis by impairing templated mutation.

Discussion

Gene conversion at the chicken Ig loci uses an array of upstream ΨVdonors as templates for homology-directed repair of lesions targeted tothe rearranged and transcribed V genes. This example shows that, inchicken B cells carrying out active Ig gene conversion, chromatin withinthe donor ΨVλ array is characterized by enrichment of acetylated H3 andH4, modifications that correlate with an open chromatin structure. Wedirectly demonstrated the importance of permissive chromatin structurefor Ig gene conversion by showing that tethering the heterochromatinprotein HP1 to the ΨVλ donor array caused local changes in chromatinstructure, diminishing the AcH3, AcH4 and diMeK4(H3) modificationscharacteristic of open chromatin. Although these changes were notaccompanied by the Me-K9 (H3) modification characteristic of closedchromatin, they caused the region to adopt a state less permissive forgene conversion. Tethering of HP1 was accompanied by a dramatic shift inthe Ig Vλ mutation spectrum, so that templated mutations were in theminority and point mutations predominated. Importantly, this effect onmutagenesis was correlated with a change in chromatin structure and notchanges in expression of the locus. Thus, chromatin structure candictate whether gene conversion occurs at a endogenously generated DNAlesion.

The Mechanism of Gene Conversion within a Complex Chromatin Landscape

Gene conversion at VA results from priming of new DNA synthesis at the3′ end of a break using a ΨVλ region as template. Gene conversionrequires synapsis between the donor and recipient DNA, as well as accessto the donor by factors that carry out homology-directed repair. Theelevated levels of H3 and H4 acetylation characteristic of the ΨVλ arrayin wild type DT40 are evidence of a relaxed chromatin structure, whichwould increase the accessibility of the ΨVλ genes to trans-actingfactors and also create a three-dimensional architecture that isfavorable for sequence synapsis.

HP1 tethered within the ΨVλ donor array impaired gene conversion at therearranged Vλ_(R), without affecting VA gene expression. Chromatinchanges caused by tethered HP1 may impair gene conversion by impedingaccess of repair factors and the invading strand to the donor template.Tethered HP1 may also contribute to larger chromosomal architecture thataffects the mechanics of DNA repair pathways, such as looping necessaryto juxtapose donor and recipient sequences. The point mutations thataccumulated in LacI-HP1 transfectants are typical of thwartedrecombinational repair, and are characteristic of cells lacking eithertrans-acting factors essential for recombination [49-55] or some or allof the ΨV donor array [56]. HP1 regulates chromatin structure andheterochromatic gene silencing in two ways, by partnering with a histonemethyltransferase [66] and by recruiting histone deacetylases [60].Tethered HP1 caused modifications characteristic of a nonpermissivechromatin structure within ΨVλ.

Histone acetylation has been documented at actively transcribedmammalian Ig genes undergoing somatic hypermutation and class switchrecombination, but whether hyperacetylation contributes to targeting ofdiversification has yet to be resolved [72-76]. A connection betweenhistone acetylation and gene conversion was suggested by experimentsshowing that treatment of DT40 cells with the histone deacetylaseinhibitor, trichostatin A, promotes genomewide histone deacetylationaccompanied by increased gene conversion at Vλ_(R)[77]. However, theinterpretation of those results is complicated by the fact that theeffects of trichostatin A are genomewide, and not specific. The DT40PolyLacO-λ_(R) cell line permits local manipulation of chromatinstructure, avoiding that complication. Moreover, we were able todemonstrate that the effects of tethering a LacI-H P1 fusion proteinwere largely reversed upon culture with IPTG, so an active mechanismmust determine chromatin modification at ΨVλ.

For studies of homologous recombination, the DT40 PolyLacO-λ_(R) B cellline has the further advantage that Ig gene conversion is aphysiological process within an endogenous locus, avoiding the need fora transgene reporter.

Chromatin Structure, Genome Stability, Aging and Gene Therapy

The importance of chromatin structure to the outcome of homologousrecombination has implications for understanding mechanisms thatnormally maintain genomic stability. There are vast numbers ofrepetitive elements distributed throughout the vertebrate genome, andrecombination between these elements can lead to genomic instability[78]. In the human genome, there are approximately one million Aluelements, and recombination between Alu elements can cause duplicationsleading to tumorigenesis and genetic disease [79,80]. Histones carryingrepressive modifications are enriched at repetitive elements [81]. Thesemodifications undoubtedly maintain transcriptional repression; ourresults suggest they may also contribute to suppression ofrecombination.

Loss of heterozygosity (LOH) occurs as a result of unequal mitoticrecombination between homologs at allelic sites. The mechanism of LOH isof particular interest, because it contributes to loss of tumorsuppressor gene function leading to tumorigenesis [82]. Recentexperiments have demonstrated an age-dependent increase in LOH in S.cerevisiae [83] and in reporter genes in Drosophila germ cells [84]; andan increase in homologous recombination in mouse pancreatic cells [85].Mechanisms proposed to explain age-associated LOH include elevated ratesof DNA damage, changes in the cell cycle distribution, and inactivationof homology-independent repair pathways with aging. The results suggestanother possibility, that relaxation of chromatin structure mayaccompany aging and promote a genome-wide increase in homologousrecombination in aging cells. This possibility is supported by recentanalysis of Drosophila [86], as well as by recent evidence that themutation in lamin A responsible for Hutchinson-Gilford Progeria Syndromeleads to a genomewide loss of H3 methylation [87].

The finding that chromatin structure regulates homologous recombinationalso has practical ramifications. Considerable current effort isdirected toward developing strategies that harness a cell's capacity forhomology-dependent repair to promote gene therapy, by providing anintact donor gene to replace a deficient target gene [88]. The resultssuggest that permissive structure at the donor will be an importantdesign parameter in developing donor genes for therapeutic applications.

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Example 5 Gene Conversion Accelerated by Distinct Modifications ofChromatin

This example demonstrates that the efficiency of repair is determined bychromatin structure of the donor. The analysis takes advantage of acis-regulatory site (polymerized lactose operators, PolyLacO) insertedjust upstream of the transcribed and diversifying VA gene in the chickenDT40 B cell line. The data show that tethering either the activator,VP16, or the histone chaperone, H IRA, alters local chromatin structureand accelerates gene conversion approximately 10-fold. While these twofactors have comparable functional outcomes, they have distinct effectson chromatin structure. VP16 tethering increases local levels ofacetylated histones H3 and H4; while HIRA tethering increases nucleosomedensity. Thus, comparable functional outcomes can be achieved bydistinct chromatin modifications.

sMaterials and Methods

Chromatin immunoprecipitation (ChIP). ChIP was carried out as previouslydescribed (Cummings et al., 2007). For all experiments at least twochromatin preparations from at least two independent stably-transfectedlines were analyzed. Figures present one representative experiment inwhich results from analysis of four separate amplifications were used tocalculate a standard deviation. Four separate amplifications of serialdilutions of template DNA were carried out, to establish that themeasured product intensities were within the linear range. Enrichment ofthe experimental amplicon was normalized to enrichment of an internalcontrol amplicon from the ovalbumin (Ova) gene, amplified in the sametube by duplex PCR; and enrichment upon ChIP with specific antibodieswas normalized to parallel experiments in which ChIP was carried outwith total input DNA controls. Inclusion of the Ova internal controlamplicon enabled us to normalize for IP efficiency, backgroundcarryover, and differences in gel loading.Enrichment=[(ΨVλ/Ova)Ab]/[ΨVλ/Ova)Input]. As an additional control, theratio of the experimental and control amplicons in the total inputcontrol was compared to a control ChIP with polyspecific IgG; in allcases, enrichment in input and IgG controls were essentially equal. Dataare presented for representative experiments; standard deviations werecalculated from four separate amplifications of serial dilutions oftemplate DNA.

Antibodies used were: anti H3 CT-pan, anti-AcH3 (06-599), anti-AcH4(06-866), and dimethylated H3(K4) (07-030) from Upstate (Lake Placid,N.Y.).

PCR primers for ChIP were:

Vλ_(R): (SEQ ID NOS: 5-28, respectively).5′-GCCGTCACTGATTGCCGTTTTCTCCCCTC-3′ and5′-CGAGACGAGGTCAGCGACTCACCTAGGAC-3′; promoter region between ψVλ1 andVλ: 5′-CTGTGGCCTGTCAGTGCTTA-3′ and 5′-GCAGGGAACCACAAGAACAT-3′; ψVλ1:5′-GGGACTTGTGTCACCAGGAT-3′ and 5′-CGCAGTCACATGTGGAATATC-3′; ψVλ5:5′-GAGCCCCATTTTCTCTCCTC-3′ and 5′-GAGATGTGCAGCAACAAGGA-3′; ψVλ13:5′-CCCTCTCCCTATGCAGGTTC-3′ and 5′-CCCCTATCACCATACCAGGA-3′; ψVλ18:5′-CCATTTTCTCCCCTCTCTCC-3′ and 5′-TCACCCTACAGCTTCAGTGC-3′; ψVλ24:5′-CCATTTTCTCCCCTCTCTCC-3′ and 5′-CAGCCCATCACTCCCTCTTA-3′; ψVλ25:5′-TCTGTTGGTTTCAGCACAGC-3′ and 5′-GCAGTTCTGTGGGATGAGGT-3′; ψVλ upstreamflank: 5′-GGCTCCTGTAGCTGATCCTG-3′ and 5′-GTTCTTTGCTCTTCGGTTGC-3′; ψVλ17at the PolyLacO-targeted allele: 5′-TAGATAGGGATAACAGGGTAATAGC-3′ and5′-AGGGCTGTACCTCAGTTTCAC-3′; OVA: 5′-ATTGCGCATTGTTATCCACA-3′ and5′-TAAGCCCTGCCAGTTCTCAT-3′; polε: 5′-GGGCTGGCTCATCAACAT-3′ and5′-CTGGGTGGCCACATAGAAGT-3′.

MNase digestion and Southern blotting. Nuclei were prepared and digestswere performed with 0, 3, 7, 15, 30, and 60 units of MNase as described(Prioleau et al., 1999). Following MNase digestion, DNA was extractedthree times with phenol:chloroform:isoamyl alcohol, and precipitatedwith ethanol. Twenty micrograms of DNA were loaded on a gel, resolved byelectrophoresis, and transferred for Southern hybridization. The lacOprobe was labeled by random priming, using as template a fragmentapproximately 500 by in length containing polyLacO repeats.

Fluorescence imaging. For fluorescence imaging, cells (2×10⁵) werecytospun onto glass slides and fixed with 2% paraformaldehyde for 15min. To visualize the nucleus, cells were stained with DAPI (Sigma,Saint Louis, Mo.). Fluorescent images were acquired using theDeltaVision microscopy system (Applied Precision) and processed withsoftWoRx software (Applied Precision).

RT-PCR. RNA was harvested from cells using TRIzol Reagent (Invitrogen).Vλ and β-actin transcripts were amplified following dilution of thetemplate. The primers for amplification of Vλ were5′-GTCAGCAAACCCAGGAGAAAC-3′ (SEQ ID NO: 3) and5′-AATCCACAGTCACTGGGCTG-3′ (SEQ ID NO: 4). The primers for amplificationof β-actin have been described (Arakawa et al., 2002).

Quantitation of slgM loss variants and sequence analysis. The slgM lossvariant assay, which measures the accumulated slgM-loss variantsresulting from frameshift or nonsense mutations in mutated V regions,was used to quantitate Ig V region diversification (Sale et al., 2001;Yabuki et al., 2005; Cummings et al., 2007). In brief, sIgM⁺ cells wereisolated by flow cytometry followed by limiting dilution cloning, andexpanded for 4 weeks. To quantitate the fraction of sIgM⁻ cells,approximately 1×10⁶ cells were stained with anti-chicken IgM-RPE(Southern Biotechnology Associates, Birmingham, Ala.), and analyzed on aFACScan with CellQuest software (BD Biosciences).

Single-cell PCR and sequence analysis were performed as described(Yabuki et al., 2005; Cummings et al., 2007). In brief, sIgM⁻ cells weresorted, aliquoted to single wells, Vλ regions amplified and sequenced,and their sequences compared to the ΨVλ donors to determine if mutationswere templated or nontemplated. The criterion for a templated mutationwas that nine consecutive bases must be an exact match in donor andrecipient. Sequences derived from two independently transfected lines.Only unique sequences were included for classification of the mutations.

Results Tethered VP16 Accelerates Gene Conversion

If repressive donor chromatin structure impairs gene conversion,activating modifications might promote gene conversion. To test thispossibility, we took advantage of the DT40 PolyLacO-λ cell lineconstructed by our laboratory. In this cell line, polymerized lactoseoperator (PolyLacO) has been inserted into the ΨVλ array betweenΨVλ17-ΨVλ20, 17 kb upstream of the expressed Vλ gene (FIG. 16A; Example4). This allows us to assay the effects of tethered regulatory factorsexpressed as fusions with lactose repressor. Control experiments haveshown that the PolyLacO tag does not affect cell proliferation, cellcycle profile, or Ig gene diversification.

Loss of histone acetylation within the ΨVλ donors correlates withdiminished gene conversion at Vλ (Example 4). We therefore tested theeffect of VP16, a potent transactivator derived from herpesvirus, whichhas been associated with the relaxation of chromatin and recruitment ofhistone acetyltransferases (Tumbar et al., 1999; Carpenter et al.,2005). We generated DT40 PolyLacO-λ_(R) transfectants stably expressingGFP-LacI-VP16. Western blotting confirmed protein expression (FIG. 16B).Cell cycle profile (FIG. 16C) and levels of λ transcription (FIG. 16D)were unaltered relative to control DT40 PolyLacO-λ GFP-LacItransfectants. Fluorescent imaging of the DT40 PolyLacO-λ GFP-LacI-VP16transfectants showed a single green spot within the nucleus, evidence ofGFP-LacI-VP16 expression and binding to PolyLacO (FIG. 16E).

We assayed histone acetylation in PolyLacO-λ GFP-LacI-VP16 and controlDT40 PolyLacO-λ GFP-LacI cells. The ΨVλ array was assayed byamplification with primers specific for seven sites, including thepromoter region between ΨVλ1 and the Vλ gene (Vλpro), ΨVλ1, ΨVλ5, ΨVλ13,ΨVλ24, ΨVλ25. (Due to the absence of polymorphisms, the ΨVλ arrays atthe two IgAλ alleles in DT40 cannot be readily distinguished by PCR.).Additionally, we assayed chromatin modifications at: (1) ΨVλ17, whichdue to a sequence polymorphism created during construction of DT40PolyLacO-λ, is the only site in the ΨVλ array at which the rearrangedand unrearranged alleles can be distinguished by use of specific PCRprimers, (2) the rearranged allele of Vλ (Vλ_(R)), (3) the unrearrangedallele of Vλ (Vλ_(U)), and (4) the Pol ε gene, an intrachromosomalcontrol. Amplification of ωVλ sites was performed in duplex with thenonexpressed ovalbumin gene (Ova). Thus, Ova functions as an internalcontrol, and the ΨVλ:Ova enrichment ratio from immunoprecipitations wasnormalized to ΨVλ:Ova ratio from total input DNA (see Material andMethods). Tethered VP16 increased both AcH3 (FIG. 17A) and AcH4 (FIG.17B) levels in DT40 PolyLacO-λGFP-LacI-VP16 transfectants relative tocontrol DT40 PolyLacO-λ GFP-LacI cells.

We used the sIgM loss variant assay to ask if tethering VP16 altered theclonal rate of Vλ sequence diversification. This fluctuation assaymeasures the fraction of variant cells that no longer expressstructurally intact sIgM, and thus scores mutation events resulting fromgene conversion, point mutation, insertion or deletion (Sale et al.,2001; Yabuki et al., 2005). While gene conversion is the predominantpathway of Ig gene diversification in chicken B cells, if geneconversion is impaired (for example by the absence of essentialrecombination factors (Sale et al., 2001; Niedzwiedz et al., 2004;Hatanaka et al., 2005; Kawamoto et al., 2005; Mcllwraith et al., 2005;Yamamoto et al., 2005), other repair outcomes, particularly pointmutations, will predominate. Thus diversification is monitored best bythis loss of function assay, rather than an sIgM− to sIgM+ gain offunction assay, which scores only gene conversion (Saribasak et al.,2006).

Independent clonal derivatives of DT40 PolyLacO-λ GFP-LacI and DT40PolyLacO-λ GFP-LacI-VP16 transfectants were established by limitingdilution cloning of sIgM⁺ cells, and following 4 weeks of culture thefraction of sIgM⁻ cells in each population determined by flow cytometryof cells stained with anti-IgM antibody. Comparison of medianpercentages of sIgM− cells showed that DT40 PolyLacO-λ GFP-LacI-VP16transfectants exhibited an 8.4-fold increase in diversification relativeto DT40 PolyLacO-λ GFP-LacI control cells (FIG. 17B). Sequence analysisof Vλ regions amplified by single cell PCR showed that mostdiversification was by gene conversion in both control cells and cellsin which VP16 was tethered at Igλ (FIG. 22).

Histone H3.3 is Enriched at the ΨVλ Donor Genes

Not only histone modifications but also localized changes in the histonecomposition of nucleosomes can contribute to regulation of chromatinstructure. One histone variant associated with chromatin activation anddeposited independently of replication is H3.3. Histone H3.3 is theprincipal H3-like variant to contain activation-associatedposttranslational modifications (McKittrick et al., 2004; Henikoff,2008), so it was of interest to ask if H3.3 was enriched at the ΨVλarray. To distinguish H3 from H3.3 by ChIP, we generated a DT40derivative which stably expresses FLAG-tagged H3.3 (H3.3-FLAG).

Characterization of H3.3 deposition by ChIP with an anti-FLAG antibodydemonstrated enrichment of the H3.3 variant at the expressed Vλ_(R)gene. Chromatin structure within the ΨVλ array was assayed byamplification of sites upstream of the rearranged and expressed Vλ geneincluding ΨVλ1, ΨVλ5, ΨVλ13, ΨVλ24, ΨVλ25, ΨVλ17_(R), Vλ_(R) (FIG. 18A),as well as the rearranged allele, Vλ_(U); and at another gene on thesame chromosome as Igλ, Pol ε. In a typical experiment, H3.3 wasenriched more than 4-fold relative to input DNA at Vλ_(R); but,strikingly, we observed considerable enrichment of H3.3 throughout theΨVλ array, with greatest enrichment at ΨVλ5 and Vλpro (FIG. 18B).Enrichment of H3.3 within the ΨVλ array does not simply represent agraded spreading of the variant from the transcribed Vλ_(R) gene tosites upstream, as proximal sites did not consistently display higherlevels of enrichment than distal sites. The non-uniform chromatinstructure of the locus suggests the presence of cis-elements,particularly in the ΨVλ5 region, that may regulate expression and/ordiversification of the locus.

Tethered HIRA Causes a Local Increase in Histone Deposition

HIRA is a histone chaperone, and one of its functions is to assemblenucleosomes containing the histone variant, H3.3 (Ray-Gallet et al.,2002; Tagami et al., 2004). Enrichment of H3.3 at the ωVλ array thussuggested that tethering HIRA might accelerate Ig gene diversification,analogous to the effect of tethered VP16. To test this, we generatedstable DT40 PolyLacO-λ HIRA-LacI transfectants, verified comparablelevels protein expression by Western blotting (FIG. 19A), and showedthat tethering did not affect cell cycle profile (FIG. 19B) or λtranscript levels (FIG. 19C). ChIP assays of AcH3 and AcH4 levels showedno difference between cells in which HIRA or GFP was tethered at Igλ.

To ask if tethered HIRA increased H3.3 levels, we generated DT40PolyLacO-λ FLAG-H3.3 HIRA-LacI transfectants, and then determinedenrichment of FLAG-H3.3 at ωVλ17_(R), where a polymorphism enablesanalysis of the tethered Igλ only. Those experiments showed a modest(1.4-fold) enrichment of FLAG-H3.3 in each of two independentlyHIRA-LacI transfectants FIG. 20A), relative to a control line. Moreover,a comparable enrichment was evident when pan-H3 antibodies were used forChIP (which detect H3 and other variants, including H3.3).

Thus, HIRA appeared to cause a net enrichment of histones, includingH3.3, and thus affected nucleosome density at ΨVλ.

Tethered HIRA Promotes Ig Gene Conversion

To ask if tethered HIRA affects the clonal rate of Ig genediversification, we established independent clonal derivatives of DT40PolyLacO-λ HIRA-LacI transfectants by limiting dilution cloning of sIgM⁺cells, and following 4 weeks of culture the fraction of sIgM⁻ cells ineach population was determined. The fraction of sIgM− cells was clearlygreater in the HIRA-LacI transfectants than in control cells (e.g. FIG.20B). Comparison of median percentages of sIgM− cells showed that DT40PolyLacO-λ HIRA-LacI cells exhibited an 11-fold increase indiversification relative to DT40 PolyLacO-λ GFP-LacI control cells (FIG.20C). This did not simply reflect overexpression of HIRA, as only amodest acceleration in diversification (1.7-fold) was evident intransfectants of DT40 stably expressing HIRA (FIG. 20C). Sequenceanalysis of Vλ regions amplified by single cell PCR showed that mostdiversification was by gene conversion in control cells and in cells inwhich HIRA was tethered at Igλ (FIG. 23).

The Fraction of Short-tract Templated Mutations is Increased by TetheredVP16 or HIRA

Tethered VP16 and HIRA accelerated the clonal rate of genediversification comparably (8.4-fold and 11-fold, respectively), andgene conversion predominated in both. To establish whether subtledifferences might distinguish mutational spectra, independent mutationalevents in the DT40 PolyLacO-λ HIRA-LacI and DT40 PolyLacO-λGFP-LacI-VP16 transfectants were compared with DT40 PolyLacO-λ GFP-LacIcontrol transfectants. Sequence changes were categorized as geneconversion if they were within a tract which was an exact match to atleast 9 by of a donor ΨVλ sequence, and contained one, two or moresingle base changes from the germline sequence. Events containing asingle base difference and two or more differences were talliedseparately as “short” and “long” tract gene conversion, because somemutations in the former class could in principle arise from pointmutations which coincidentally matched ωVλ donor sequences. Nontemplatedevents, consisting of point mutations, deletions, and insertions, werealso scored.

In DT40 PolyLacO-λ GFP-LacI-VP16 transfectants, 74% of events weretemplated, but a significant fraction of templated events (39%, or 29%of all mutation events) were short tract rather than long-tract geneconversion (Table 1, FIG. 22). There was also a significant fractions ofinsertions (13% compared to 0%). Gene conversion events similarlypredominated (87%) among the events analyzed in from DT40 PolyLacO-λ_(R)HIRA-LacI cells (Table 1, FIG. 23). Most gene conversion events werelong-tract, but a significant minority (24%, or 21% of all mutationevents) were short tract gene conversion. The remainder of events werepoint mutations (11%) and insertions (2%). We previously characterizedmutagenic events in control DT40 PolyLacO-λ GFP-LacI transfectants, andfound that most mutations were due to long-tract gene conversion events(77%), while a small fraction (3%) were either short tract conversionevents or point mutations that coincidentally matched a donor sequence(Example 4: summarized in Table 1). The remaining mutations (20%) failedto correspond to any of the pseudogene donors and were, therefore, clearpoint mutations.

TABLE 1 Effect of tethered HP1 and VP16 on gene conversion. Summary ofsequences of Vλ regions carrying unique mutations from DT40 PolyLacO-λGFP-Lacl (n = 71; (Example 4), DT40 PolyLacO-λ GFP-Lacl-VP16 (n = 31),and DT40 PolyLacO-λ HIRA-Lacl (n = 137) transfectants. Data derived fromat least two independent transfections. GFP- Gene Conversion GFP-LaclLacl-VP16 HIRA-Lacl Long (2 or 77% 45% 66% more nt) Short (1 nt) 3% 29%21% Total 80% 74% 87% Point mutation 20% 13% 11% Insertion 0 13% 2%

Thus, acceleration of diversification by tethered VP16 and HIRA is dueto increased levels of templated mutation. In both cases, most templatedtracts contained two or more mutations; but about one-third of tractscontained only a single mutation. In contrast, essentially all geneconversion events in the GFP-LacI control cells contained at least twomutations. This suggests that the changes in donor chromatin structuremay not only accelerate gene conversion but also limit the extent ofgene conversion tracts; or that accelerated gene conversion may titratefactors that contribute to extending the length of conversion tracts.

Tethered HIRA Increases Nucleosome Density

The results above show that tethered VP16 and HIRA had comparableeffects on the outcome of Ig gene diversification, although each haddifferent local effects: VP16 increased AcH3 and AcH4, while HIRAincreased H3.3 and overall histone loading. To establish how loading ofthese different factors might affect chromatin, we probed chromatinstructure using micrococcal nuclease (MNase). Nuclei were harvested,treated with varied amounts of MNase, genomic DNA purified, resolved andblotted, and then probed with labeled DNA homologous to the LacOrepeats. The MNase digestion pattern of DT40 PolyLacO-λ GFP-LacI andDT40 PolyLacO-λ GFP-LacI-VP16 cells appeared comparable by this assay(FIG. 21). Digestion with high levels of MNase produced a clear limitproduct. This is the region of DNA (about 150 bp) that contacts thenucleosome, and is thereby protected from MNase. Spacing betweenindividual nucleosomes can vary, causing slight variation in sizes ofnucleosome multimers produced upon partial digestion, and producing thecharacteristically blurred bands migrating more slowly in the gel.

In contrast, MNase digestion of chromatin from DT40 PolyLacO HIRA-LacIcells revealed a different pattern: distinctive laddering was observed,not found in the control (FIG. 21, right). The sharply defined series ofdigestion products spanned from the 1-mers produced upon limit digestionup to 8-mers and beyond; while the ladder became blurred at sizes largerthan 4-mers in the control and VP16-expressing cells. This suggests thattethering the histone chaperone HIRA increased the uniformity of linkersize. That would occur if local nucleosome density were increased,consistent with the known role of HIRA as a histone chaperone, as wellas with the evidence that tethering HIRA increased local enrichment ofH3.3 and H3 (FIG. 18C).

Discussion

This example has shown that gene conversion can be accelerated bychanges in chromatin structure, and that there are at least twodifferent means to this end. Gene conversion was accelerated by an orderof magnitude upon tethering either the activator, VP16,or the histonechaperone, HIRA, at the ΨVλ array in the chicken DT40 B cell line.Tethering VP16 increased levels of AcH3 and AcH4; while tethering HIRAdid not affect those chromatin marks, but increased nucleosome density,in part by loading H3.3. The fact that different changes in the state ofchromatin are similarly enabling for homologous recombination shows thatthe cell has available redundant pathways for regulating uses of DNA inrecombination.

The effect of tethering VP16 and HIRA on Ig gene diversification isdistinct from that of tethering the heterochromatin protein HP1.Tethering HP1 caused a nearly 3-fold increase in point mutations,accompanied by a decrease in templated mutations (Example 4). Thesedifferences show that donor chromatin structure may be either activatedor repressed for homology-directed DNA repair, with consequences on therepair outcome.

Currently, allelic homology-directed repair is thought to predominate inS phase, and to depend on sister chromatids for templates. The potentialfor local alterations of chromatin structure to activate regions forhomology-directed repair suggests that regulation may be more complex,and require not only the presence of a homologous donor but alsochromatin structure that is conducive to repair.

Tethering of HIRA or VP16 accelerated clonal diversification rates bypromoting templated mutation. In both cases, an increase was evident inthe fraction of templated mutations in which only a single templatedbase change was evident in the mutation tract. While these “short tract”mutations comprised only 29% and 21% of total mutations in VP16 and HIR1transfectants, respectively, this category of mutations was essentiallyabsent (3%) in GFP-LacI transfectants.

Short gene conversion tracts could arise if factors necessary to promotelong tract gene conversion are not sufficiently abundant to support theincrease in gene in cells in which either VP16 or HIRA is tethered toPolyLacO. Alternatively, the increase in mutations in this categorycould reflect changes in chromatin structure caused by tethering thesefactors, which could affect both nucleosome position and linker length.

Nucleosome density is increased upon tethering HIRA. This densityincrease is likely to reflect diminished linker length. If linkerregions are preferred templates for gene conversion, shortening of thoseregions in the HIRA transfectants may contribute to decreased geneconversion tract length. Shortening of nucleosome length may,alternatively, re-phase nucleosomes so that, although chromatin isactive for recombination, the nonhomologous regions between the ΨVbecome less accessible .

The role of HIRA at the pseudogenes is a novel example of how histoneloading affects DNA repair. The role of HIRA at the untranscribedpseudogenes is particularly interesting as many studies have foundtranscription required for de novo H3.3 deposition (Ahmad and Henikoff,2002a; Ahmad and Henikoff, 2002b; Janicki et al., 2004; Wirbelauer etal., 2005). The results now suggest that transcription is notprerequisite to H3.3 loading.

These results increase our understanding of the constraints exerted bychromatin constraints on recombination. In addition, they provideinsight into the necessary chromatinization of molecules used for genetargeting and/or gene therapy.

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Cummings, W. J., et al. (2007). See Example 4.

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Example 6 Generation of Diagnostic Antibodies to Mesothelin and HE4

This example illustrates how one can use the invention to developantibodies against mesothelin and HE4, well-validated ovarian cancerbiomarkers. The antibodies identified will be valuable new diagnosticreagents, adding useful redundancy to clinical tests. This examplefurther validates the utility of DT40 PolyLacO as a platform forselection of diagnostic antibodies that recognize other biomarkers.

DT40 PolyLacO as a vehicle for antibody development: identification ofnew antibodies against two ovarian cancer biomarkers, mesothelin(Scholler et al., 1999; Frierson et al., 2003) and HE4 (Hellstrom etal., 2003).

Mesothelin is an epithelial differentiation marker highly expressed oncancer cells of many origins, including ovarian cancer (Robinson et al.,2003). HE4 (aka WFDC2) is a member of the whey-acidic protein (WAP)family, members of which are secreted at high levels and associated withcancer, although function is not understood (Bouchard et al., 2006). Wehave obtained recombinant mesothelin and HE4. These proteins areexpressed in yeast as biotin fusions, which facilitates binding tostreptavidin-coupled beads or plates for selection or ELISA assays(Scholler et al., 2006; Bergan et at., 2007). We have also obtainedsingle-chain antibodies (scFv) against mesothelin and HE4 (Scholler etal., 2008), which provide a standard for initial comparison ofaffinities of newly identified antibodies.

To identify high affinity antibodies to mesothelin and HE4, iterativehypermutation and clonal selection for recognition of each recombinantantigen is performed. Clones are expanded under conditions thataccelerate hypermutation; selected for clones that bind mesothelin orHE4 with high affinity by magnetic activated cell sorting; and antibodyaffinity of supernatants is assayed by ELISA assay. Populationsproducing high affinity antibodies are further selected to enhanceaffinity and specificity (FIG. 3).

Selection can be initiated on three populations of 10⁸ cells each(corresponding to 100 ml of culture). Each population can be derivedfrom a single cell isolated by limiting dilution, and then culturedunder conditions that accelerate hypermutation. To minimize enrichmentof “sticky” cells, which bind beads alone, prior for enrichment for adesired specificity each population is first cleared with biotin-boundbeads. Enrichment for a desired specificity is achieved conveniently bymagnetic activated cell sorting (MACS). Using MACS, it is possible toenrich very quickly from very large sample sizes (10⁸ cells),consistently achieving 100-fold enrichment of populations comprising<0.1% of the starting sample (Volna et al., 2007). The selectedpopulation is expanded and reselected using the same protocol.

Based on results of others (e.g. Cumbers et al., 2002) we predict thatcells producing antibodies specific for each “antigen” will be recoveredat or before round 2-3 of iterative hypermutation and selection (2-3weeks), and probably earlier. One can test antibody affinity of cellpools by ELISA assay; and when affinity is in the 1-10 nM range,limiting dilution cloning is performed, and clones that make highaffinity antibody identified. Limiting dilution cloning is carried outin medium containing IPTG, which releases repressor from operator todecelerate hypermutation. Antibody produced from these clonalpopulations will be further characterized, as described below.

Three criteria can be used to establish that this approach hassucceeded:

1. Affinity and specificity. Efficacy of antibody development isvalidated by demonstrating that antibody affinity increases in thecourse of selection. Antibody affinities are first measured by ELISAassays of dilutions of supernatant of cells cultured for 48 hr, in thepresence of IPTG, to minimize further hypermutation, in IgM-depletedmedium, to minimize background in the ELISA. To ensure that antibodiesrecognize the antigen and not the recombinant scaffold in which it isdisplayed, reactivity is tested with biotin and streptavidin. Aside-by-side comparison of anti-mesothelin and anti-HE4 antibodiesagainst both recombinant antigens serves as a test of cross-reactivity.Affinities are compared to control scFv antibodies directed tomesothelin or HE4. Following establishment of high affinity clonalpopulations, Kd will be determined quantitatively by surface plasmonresonance by Biacore.

2. Targeting of mutagenesis. The cellular mechanism that hypermutatesantibody genes in the course of clonal selection concentrates mutationsin the CDRs, the subdomains of the V regions that contact antigen. Wepredict that high affinity clones isolated by accelerated hypermutationand selection will similarly concentrate mutations in the CDRs. To testthis, one can use single-cell PCR to amplify the VH and VL regions ofindividual B cells following affinity selection, sequence the VH and VLregions, and compare these sequences with those of the unselectedpopulation.

3. Maintenance of specificity and affinity upon culture with IPTG. Thepower of this platform for antibody production depends in part on theability to limit hypermutation when a high affinity clone has beengenerated. To test this, one can culture clonal populations in thepresence and absence of IPTG, and compare average affinities of expandedcultures by flow cytometry of cells bound to fluorescent-taggedmesothelin or HE4. Affinities are predicted to remain constant incultures containing IPTG, but to increase or decrease in cultureslacking IPTG. Thus, the distribution of fluorescence signal will bebroader in the IPTG-containing cultures.

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Bouchard, D., et al. (2006). Lancet Oncol 7, 167-174.

Cumbers, S. J., et al. (2002). Nat Biotechnol 20, 1129-1134.

Frierson, H. F., Jr., et al. (2003). Hum Pathol 34, 605-609.

Hellstrom, I., et al. (2003). Cancer Res 63, 3695-3700.

Hung, C. F., et al. (2008). Immunol Rev 222, 43-69.

Liu, X. Y., et al. (2008). Immunol Rev 222, 9-27.

Robinson, B. W., et al. (2003). Lancet 362, 1612-1616.

Sale, J. E. (2004). DNA Repair (Amst) 3, 693-702.

Scholler, N., et al. (1999). Proc Natl Acad Sci U S A 96, 11531-11536.

Scholler, N., et al. (2006). J Immunol Methods 317, 132-143.

Scholler, N., et al. (2008). Clin Cancer Res 14, 2647-2655.

Seo, H., et al. (2005). Nat Biotechnol 23, 731-735.

Volna, P., et al. (2007). Nucleic Acids Res 35, 2748-2758.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to describemore fully the state of the art to which this invention pertains.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A B cell modified to reversibly induce diversification of a targetgene, the B cell comprising: (a) a cis-regulatory element operablylinked to a target gene, wherein the target gene comprises a promoterand a coding region; (b) a fusion construct that encodes a tetheringfactor fused to a diversification factor, wherein the tethering factorspecifically binds to the cis-regulatory element.
 2. The B cell of claim1, which is a DT40 chicken B cell.
 3. The B cell of claim 1, which is ahuman B cell.
 4. The B cell of claim 1, wherein the cis-regulatoryelement is a polymerized Lactose operator (LacO) and the tetheringfactor is a lactose repressor (LacI).
 5. The B cell of claim 1, whereinthe cis-regulatory element is a tetracycline operator (TetO) and thetethering factor is tetracycline repressor (TetR).
 6. The B cell ofclaim 1, wherein the diversification factor is a transcriptionalregulator, a heterochromatin-associated protein, a histone chaperone, achromatin remodeler, a component of the nuclear pore complex, a generegulator or a combination thereof.
 7. The B cell of claim 1, whereinthe diversification factor is a DNA repair factor, a DNA replicationfactor, a resolvase, a helicase, a cell cycle regulator, a ubquitylationfactor, a sumoylation factor, or a combination thereof.
 8. The B cell ofclaim 6, wherein the transcriptional regulator is VP16 or E47.
 9. The Bcell of claim 6, wherein the heterochromatin-associated protein is HP1.10. The B cell of claim 6, wherein the histone chaperone is HIRA. 11.The B cell of claim 1, wherein the target gene is a human Ig gene. 12.The B cell of claim 1, wherein the target gene is an IgL gene.
 13. The Bcell of claim 1, wherein the target gene is an IgH gene.
 14. The B cellof claim 1, wherein the target gene comprises a heterologous codingregion and regions encoding a transmembrane domain and a cytoplasmictail sufficient to effect display of the target gene product on the Bcell surface.
 15. A method of producing a repertoire of polypeptideshaving variant sequences of a polypeptide of interest, the methodcomprising: (a) culturing the B cell of claim 1 in conditions that allowexpression of the diversification factor, wherein the target gene of theB cell contains the coding region of the polypeptide of interest,thereby permitting diversification of the coding region; and (b)maintaining the culture under conditions that permit proliferation ofthe B cell until a plurality of B cells and the desired repertoire isobtained.
 16. A method of producing B cells that produce an optimizedpolypeptide of interest, the method comprising: (a) culturing a B cellof claim 1 in conditions that allow expression of the diversificationfactor, wherein the target gene of the B cell contains the coding regionof the polypeptide of interest, and wherein and the B cell expresses thepolypeptide of interest on the surface of the B cell; (b) selectingcells from the culture that bind a ligand that specifically binds thepolypeptide of interest expressed on the B cell surface; and (c)repeating steps (a) and (b) until cells are selected that have a desiredaffinity for the ligand that specifically binds the polypeptide ofinterest.
 17. The method of claim 15, wherein the polypeptide ofinterest is an Ig.
 18. The method of claim 17, wherein the Ig is an IgL,IgH or both.
 19. The method of claim 15, further comprising adding aregulatory molecule to the culture, wherein the regulatory moleculemodulates binding of the tethering factor to the cis-regulatory element,thereby modulating diversification of the coding region.
 20. The methodof claim 15, further comprising removing a regulatory molecule from theculture, wherein the regulatory molecule modulates binding of thetethering factor to the cis-regulatory element, thereby modulatingdiversification of the coding region.
 21. The method of claim 15,further comprising modulating expression of a regulatory gene in the Bcell, wherein the regulatory gene regulates diversification, therebymodulating diversification of the region that encodes the polypeptide ofinterest.
 22. A kit comprising: (a) a B cell comprising a cis-regulatoryelement operably linked to a target gene, wherein the target genecomprises a promoter and a coding region; and (b) one or morecontainers; and (c) one or more fusion constructs stored in a containerof (b), wherein each fusion construct is expressable in the B cell andencodes a tethering factor fused to a diversification factor, whereinthe tethering factor specifically binds to the cis-regulatory element ofthe B cell.
 23. The kit of claim 22, wherein the B cell is a DT40chicken B cell.
 24. The kit of claim 22, wherein the B cell is a human Bcell.
 25. The kit of claim 22, wherein the cis-regulatory element is apolymerized Lactose operator (LacO) and the tethering factor is alactose repressor (LacI).
 26. The kit of claim 22, wherein thecis-regulatory element is a tetracycline operator (TetO) and thetethering factor is tetracycline repressor (TetR).
 27. The kit of claim22, wherein the diversification factor is a transcriptional regulator, aheterochromatin-associated protein, a histone chaperone, a chromatinremodeler, a component of the nuclear pore complex, a gene regulator ora combination thereof.
 28. The kit of claim 22, wherein thediversification factor is a DNA repair factor, a DNA replication factor,a resolvase, a helicase, a cell cycle regulator, a ubquitylation factor,a sumoylation factor, or a combination thereof.
 29. The kit of claim 27,wherein the transcriptional regulator is VP16 or E47.
 30. The kit ofclaim 27, wherein the heterochromatin-associated protein is HP1.
 31. Thekit of claim 27, wherein the histone chaperone is HIRA.
 32. The kit ofclaim 22, wherein the target gene is a human Ig gene.
 33. The kit ofclaim 22, wherein the target gene is an IgL gene.
 34. The kit of claim22, wherein the target gene is an IgH gene.
 35. The kit of claim 22,wherein the target gene comprises a heterologous coding region andregions encoding a transmembrane domain and a cytoplasmic tailsufficient to effect display of the target gene product on the B cellsurface.